In vitro model for a tumor microenvironment

ABSTRACT

Methods for mimicking a tumor microenvironment in vitro are provided. The methods comprise indirectly applying a shear stress upon at least one tumor cell type plated on a surface within a cell culture container. Methods for mimicking tumor metastasis and methods for testing drugs or compounds in such systems are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/893,402, filed Oct. 21, 2013, the entirety ofwhich is herein incorporated by reference.

GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under Contract NumberHSN261201300024C awarded by the National Cancer Institute at theNational Institutes of Health. The Government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention generally relates to methods for mimicking a tumormicroenvironment in vitro. The present invention also relates to methodsfor testing drugs or compounds in such systems and identifying potentialcancer drug targets.

BACKGROUND OF THE INVENTION

In vivo, the tumor microenvironment is a complex milieu containingmultiple cell types including tumor cells, vascular cells such asendothelial cells, and stromal cells, such as fibroblasts. In addition,in vivo, these cells are exposed to blood flow and various biologicaltransport conditions. In vivo, microvascular cells in a tumor areaffected by blood flow and communicate with tumor and non-tumor cells,both physically and through diffusible factors. In addition, the tumorvasculature is abnormal, characterized by chaotic branching, a low flowrate, and leaky vessels, and thus serves as a major transport barrier toanticancer therapies that target tumor cells. The interplay betweentumor cells, endothelial cells, and stromal cells affects each celltype, leading to increased angiogenesis and tumor cell proliferation,and this crosstalk may be an important factor in determining theresponsiveness of tumor cells to anticancer drugs.

Conventional in vitro tumor models using static monocultures of tumorcells fail to adequately model in vivo tumor biology. Current in vitrotumor models also do not accurately predict efficacy and safety ofanticancer therapies in vivo. Traditional in vitro studies performedunder static conditions are generally poor predictors of drugsensitivity, due to the lack of representation of components of thetumor microenvironment. Furthermore, the conventional models often donot exhibit responses to drugs or compounds at concentrations thatproduce the response in vivo, instead requiring much higherconcentrations of the drug or compound to induce the same response.Thus, there exists a need in the art for methods for accuratelymimicking the in vivo tumor microenvironment in vitro. Such methodswould improve the accuracy of preclinical screening of anticancer agentsfor efficacy and safety.

SUMMARY OF THE INVENTION

A method for mimicking a tumor microenvironment in vitro is provided.The method comprises adding a culture medium to a cell culture containerand plating at least one tumor cell type on a surface within the cellculture container. A shear stress is indirectly applied upon the atleast one tumor cell type, the shear stress resulting from flow of theculture medium induced by a flow device, the flow mimicking flow towhich the tumor cells are indirectly exposed in vivo in the tumormicroenvironment. The flow is time-variant.

Another method for mimicking a tumor microenvironment in vitro is alsoprovided. The method comprising adding a culture medium to a cellculture container and plating at least one tumor cell type on a firstsurface of a porous membrane within the cell culture container. A shearstress is indirectly applied upon the at least one tumor cell type byapplying a shear stress upon a second surface of the porous membrane,the shear stress resulting from flow of the culture medium induced by aflow device, the flow mimicking flow to which the tumor cells areindirectly exposed in vivo in the tumor microenvironment.

Yet another method for mimicking a tumor microenvironment in vitro isalso provided. The method comprises adding a culture medium to a cellculture container and plating at least one tumor cell type or stromalcell type on a first surface of a porous membrane within the cellculture container. When the stromal cell type is plated on the firstsurface of the porous membrane, at least one tumor cell type is presenton a surface within the cell culture container. A shear stress isindirectly applied upon the at least one tumor cell type by applying ashear stress upon a second surface of the porous membrane, the shearstress resulting from flow of the culture medium induced by a flowdevice, the flow mimicking flow to which the tumor cells are indirectlyexposed in vivo in the tumor microenvironment.

A further method for mimicking a tumor microenvironment in vitro is alsoprovided. The method comprises adding a culture medium to a cell culturecontainer and plating at least one stromal cell type on a first surfaceof a first porous membrane within the cell culture container. A secondporous membrane is placed on the plated stromal cell type, such that afirst surface of the second porous membrane contacts the plated stromalcells. At least one tumor cell type is plated on a second surface of thesecond porous membrane. A shear stress is indirectly applied upon the atleast one tumor cell type by applying a shear stress upon the secondsurface of the first porous membrane, the shear stress resulting fromflow of the culture medium induced by a flow device, the flow mimickingflow to which the tumor cells are indirectly exposed in vivo in thetumor microenvironment.

An in vitro method of testing a drug or a compound for an effect on atumor is provided. The method comprises mimicking the tumormicroenvironment and adding a drug or a compound to the culture medium.A shear stress is indirectly applied upon the at least one tumor celltype directly or indirectly exposed to the drug or the compound. Achange in the at least one tumor cell type, in the presence of the drugor the compound, indicates that the drug or the compound has an effecton the tumor.

A method for mimicking tumor metastasis in vitro is also provided. Themethod comprises introducing cells of the at least one tumor cell typecultured according to any of the methods described above into an invitro system that models an organ or tissue.

Another method for mimicking tumor metastasis is also provided. Themethod comprises introducing cells of the at least one tumor cell typecultured according to any of the methods described above into an animal.

Yet another method for mimicking tumor metastasis in vitro is provided.The method comprises adding a culture medium to a cell culture containerand plating at least one cell type on a first surface of a porousmembrane within the cell culture container, wherein the porous membraneis suspended in the cell culture container such that the first surfaceis proximal and in spaced relation to a bottom surface of the cellculture container, thereby defining within the cell culture container alower volume comprising the at least one cell type and an upper volumecomprising a second surface of the porous membrane. A shear stress isindirectly applied upon the at least one cell type, the shear stressresulting from flow of the culture medium induced by a flow device, theflow mimicking flow to which the cells are indirectly exposed in vivo.Tumor cells derived from a human or a humanized animal are introducedinto the upper volume or the lower volume.

An in vitro method of testing a drug or a compound for an effect ontumor metastasis is provided. The method comprises mimicking tumormetastasis in vitro and adding a drug or a compound to the culturemedium. A change in the cells of the at least one tumor cell type in thein vitro system that models the organ or tissue, in the presence of thedrug or the compound, indicates that the drug or the compound has aneffect on tumor metastasis.

A method for selecting a chemotherapy regimen to be administered to asubject having a tumor is provided. The method comprises testing a drugor a compound in vitro for an effect on the tumor or testing a drug or acompound for an in vitro effect on tumor metastasis, wherein the atleast one tumor cell type comprises tumor cells derived from thesubject's tumor. The method further comprises determining whether toadminister the drug or the compound to the subject based on the resultsof the in vitro testing.

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a cone-and-plate device and indirect application of ashear stress to tumor cells.

FIG. 2 is a perspective of the clip that mounts on the cell culturecontainer and secures inflow and outflow tubing to perfuse the upper andlower volumes.

FIG. 3 shows the positioning of two clips in a cell culture container.

FIG. 4A shows a Doppler sonography image of the central bronchial arteryin a patient diagnosed with a pulmonary lesion.

FIG. 4B depicts wall shear stress calculations (dynes/cm²) of theDoppler flow signal of a human pulmonary lesion.

FIG. 4C is a schematic illustration of an exemplary arterial bloodsupply in a pulmonary lesion. “PA” stands for pulmonary artery, “cBA”stands for central bronchial artery, and “pBA” stands for peripheralbronchial artery.

FIGS. 5-10 are schematic diagrams illustrating methods for mimicking atumor microenvironment in vitro.

FIG. 11 shows an exemplary system that models the liver, which can beused to mimic tumor metastasis in vitro.

FIGS. 12A-D depict exemplary configurations for modeling tumormetastasis in vitro by pumping culture medium out of a cell culturecontainer comprising at least one tumor cell type and into an in vitrosystem that models the liver.

FIGS. 13A-C provide fluorescent microscopy images for human dermalmicrovascular endothelial cells (FIG. 13A), human fibroblasts (FIG.13B), and A549 human non-small cell carcinoma (NSCLC) tumor cells (FIG.13C) cultured using the method depicted in FIG. 9 under hemodynamicshear stress. FIGS. 13D-F provide fluorescent microscopy images for A549tumor cells cultured using the method depicted in FIG. 9 underhemodynamic shear stress in the substantial absence of exogenously addedextracellular matrix (ECM) (FIG. 13D), where the A549 tumor cells wereplated on a layer of collagen (FIG. 13E), or where the A549 tumor cellswere plated on a layer of collagen and another layer of collagen wasdeposited on top of the plated A549 tumor cells such that the collagensubstantially surrounded the tumor cells (FIG. 13F, “collagensandwich”).

FIG. 14A provides results from an assay measuring cell growth of A549tumor cells under two-dimensional static conditions or using the methoddepicted in FIG. 9.

FIG. 14B provides a schematic illustration of the qualitativedifferences in the growth rate of tumor cells cultured in statictwo-dimensional cultures (“In Vitro”), in the in vitro tumormicroenvironments described herein (“In vitro tumor microenvironment”),and in xenografts.

FIG. 14C provides results from an assay measuring the growth of A549tumor cells cultured using the method depicted in FIG. 9 underhemodynamic shear stress in the substantial absence of exogenously addedextracellular matrix (“no matrix”), where the A549 tumor cells wereplated on a single layer of collagen (“collagen layer”), or where theA549 tumor cells were plated on a layer of collagen and another layer ofcollagen was deposited on top of the plated A549 tumor cells such thatthe collagen substantially surrounded the tumor cells (“collagensandwich”).

FIG. 15 provides results from a permeability assay assessing thepermeability of endothelial cells cultured in the presence (“tumorcells”) or absence (“no tumor cells”) plated on the opposing side of aporous membrane and cultured under hemodynamic shear stress.

FIGS. 16A and 16B provide a dendogram (FIG. 16A) and a heatmap (FIG.16B) showing the expression and clustering of 14,159 genes in A549 tumorcells grown under static two-dimensional conditions (“plastic”), inxenografts, or using the method shown in FIG. 9 under hemodynamic shearstress in the substantial absence of exogenously added extracellularmatrix (“NC” or “no collagen”), where the A549 tumor cells were platedon a single layer of collagen (“CL” or “collagen layer”) or where theA549 tumor cells were plated on a layer of collagen and another layer ofcollagen was deposited on top of the plated A549 tumor cells such thatthe collagen substantially surrounded the tumor cells (“CS” or “collagensandwich”).

FIGS. 17A and 17B provide a dendogram (FIG. 17A) and a heatmap showingthe expression and clustering of 7935 genes differentially expressedbetween xenografts and static two-dimensional cultures of A549 tumorcells in A549 tumor cells grown in xenografts or using the methoddepicted in FIG. 9 under hemodynamic shear stress in the substantialabsence of exogenously added extracellular matrix (NC) or in a collagensandwich (CS), as compared to static-two dimensional cultures(“plastic”).

FIGS. 18A and 18B provide a dendogram (FIG. 18A) and a heatmap (FIG.18B) showing the clustering of 48 genes annotated with “non-small celllung cancer” in the Kyoto Encyclopedia of Genes and Genomes (KEGG)database in A549 tumor cells grown in xenografts or using the methoddepicted in FIG. 9 under hemodynamic shear stress in the substantialabsence of exogenously added extracellular matrix (NC) or in a collagensandwich (CS), as compared to static-two dimensional cultures(“plastic”).

FIGS. 19A and 19B provide results showing the inhibition of the growthof A549 tumor cells in the presence of cisplatin, MK2206, or selumetinib(AZD6244).

FIGS. 20A-F are fluorescent microscopy images of hepatocytes culturedunder static conditions or in the presence of controlled hemodynamics.

FIG. 21A is a fluorescent microscopy image of hepatocytes cultured undercontrolled hemodynamics.

FIG. 21B is a fluorescent microscopy image of in vivo liver.

FIG. 21C shows transmission electron microscopy images of hepatocytescultured under controlled hemodynamics.

FIGS. 22A-B provide data for albumin and urea secretion in hepatocytescultured under static conditions or controlled hemodynamics.

FIGS. 23A-D provide metabolic gene expression data for hepatocytescultured under static conditions or controlled hemodynamics.

FIGS. 24A-B provide cytochrome p450 activity data for hepatocytescultured under static conditions or controlled hemodynamics.

FIG. 24C is a fluorescent microscopy image from an assay for transporteractivity in hepatocytes cultured under controlled hemodynamics.

FIG. 25 shows gene expression data for an in vitro fatty liver model.

FIG. 26 shows gene expression data for an in vitro fatty liver model.

FIGS. 27A-B provide fluorescent microscopy images of hepatocytescultured under healthy conditions or conditions that mimic fatty liverdisease.

FIG. 28 shows a transmission electron microscopy image of rathepatocytes cultured under high glucose/high insulin conditions.

FIGS. 29A-B provide results from assays measuring total lipids and totaltriglycerides in hepatocytes cultured under healthy conditions orconditions that mimic fatty liver disease.

FIGS. 30A-B provide gene expression data for hepatocytes cultured underhealthy conditions or conditions that mimic fatty liver disease.

FIGS. 31A-B provide metabolic gene expression data and cytochrome p450activity data for hepatocytes cultured under healthy conditions orconditions that mimic fatty liver disease.

FIGS. 32A-3C show fluorescent microscopy images from hepatocytescultured under healthy conditions or under conditions that mimic fattyliver disease, in the presence or absence of pioglitazone.

FIG. 33 provides results from an assay measuring total triglycerides inhepatocytes cultured under healthy conditions or under conditions thatmimic fatty liver disease, in the presence or absence of pioglitazone.

FIG. 34 provides metabolic gene expression data for hepatocytes culturedunder healthy conditions or under conditions that mimic fatty liverdisease, in the presence or absence of pioglitazone.

FIGS. 35A-C provide cytochrome activity data for hepatocytes culturedunder controlled hemodynamic conditions or static conditions in thepresence of phenobarbital or rifampicin.

FIG. 36A provides fluorescence microscopy images showing the toxicityresponse of hepatocytes cultured under controlled hemodynamic conditionsto chlorpromazine at an in vivo plasma C_(max) concentration.

FIG. 36B provides data showing a toxicity dose-response for hepatocytescultured under controlled hemodynamics or static conditions and exposedto varying concentrations of chlorpromazine.

FIGS. 37A-B provides data showing upregulation of oxidativestress-related toxicity genes (FIG. 37A) and metabolic genes (FIG. 37B)in response to chlorpromazine in hepatocytes cultured under controlledhemodynamic conditions.

FIG. 38 provides acute toxicity data, measured by release of miRNA122,in hepatocytes cultured under controlled hemodynamic or staticconditions in response to chlorpromazine.

FIG. 39 provides fluorescence microscopy images showing sublethaltoxicity and cholestatic changes in hepatocytes cultured undercontrolled hemodynamic conditions in response to treatment withtroglitazone.

FIG. 40 provides data showing the upregulation of oxidativestress-related genes and MRP3 and MRP4 genes in hepatocytes culturedunder controlled hemodynamic conditions in response to treatment withtroglitazone.

FIG. 41A provides a fluorescence microscopy image showing retention ofpolarized morphology in canine hepatocytes cultured under controlledhemodynamic conditions.

FIG. 41B provides gene expression data showing expression of CYP1A1 andCYP3A1 in canine hepatocytes cultured under controlled hemodynamicconditions or static conditions.

FIG. 42 provides a fluorescence microscopy image showing retention ofpolarized morphology in hepatocytes derived from inducible pluripotentstem cells (iPSCs) cultured under controlled hemodynamic conditions.

FIGS. 43A-C provide gene expression data showing the expression ofmetabolic genes and differentiation genes in iPSC-derived hepatocytescultured under controlled hemodynamic conditions.

Corresponding reference characters indicate corresponding partsthroughout the drawings.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for mimicking a tumormicroenvironment in vitro. In contrast to the static monoculture modelscurrently used as the standard in vitro models of tumor biology by thepharmaceutical and biopharmaceutical industries, the methods of thepresent invention recreate the tumor microenvironment and can be used toassess multiple aspects of cancer, including endothelial cell barrierfunction, tumor growth, cell proliferation, cell migration, cellinvasion, and alterations in responsiveness of tumor cells to anticancertherapies.

A method for mimicking a tumor microenvironment in vitro is provided.The method comprises adding a culture medium to a cell culturecontainer, plating at least one tumor cell type on a surface within thecell culture container, and indirectly applying a shear stress upon theat least one tumor cell type. The shear stress results from flow of theculture medium induced by a flow device. The flow mimics flow to whichthe tumor cells are indirectly exposed in vivo in the tumormicroenvironment. The flow is time-variant.

At least one extracellular matrix component can be deposited on thesurface within the cell culture container, and the at least one tumorcell type can be plated on the at least one extracellular matrixcomponent. Alternatively, the at least one tumor cell type can besuspended in a solution comprising at least one extracellular matrixcomponent to create a suspension comprising the at least one tumor celltype and the at least one extracellular matrix component. The suspensioncan then be deposited on the surface within the cell culture container.The shear stress can be indirectly applied upon the at least oneextracellular matrix component and the at least one tumor cell type.

The method can further comprise plating the at least one tumor cell typeon a first surface of a porous membrane and indirectly applying theshear stress upon the at least one tumor cell type by applying the shearstress upon a second surface of the porous membrane.

Another method for mimicking a tumor microenvironment in vitro is alsoprovided. The method comprises adding a culture medium to a cell culturecontainer, plating at least one tumor cell type on a first surface of aporous membrane within the cell culture container, and indirectlyapplying a shear stress upon the at least one tumor cell type byapplying a shear stress upon a second surface of the porous membrane.The shear stress results from flow of the culture medium induced by aflow device. The flow mimics flow to which the tumor cells areindirectly exposed in vivo in the tumor microenvironment.

Yet another method for mimicking a tumor microenvironment in vitro isprovided. The method comprises adding a culture medium to a cell culturecontainer and plating at least one tumor cell type or stromal cell typeon a first surface of a porous membrane within the cell culturecontainer. When the stromal cell type is plated on the first surface ofthe porous membrane, at least one tumor cell type is present on asurface within the cell culture container. Shear stress is indirectlyapplied upon the at least one tumor cell type by applying a shear stressupon a second surface of the porous membrane, the shear stress resultingfrom flow of the culture medium induced by a flow device, the flowmimicking flow to which the tumor cells are indirectly exposed in vivoin the tumor microenvironment.

In any of the methods wherein the at least one tumor cell type is platedon a first surface of a porous membrane, the porous membrane can besuspended in the cell culture container such that the first surface isproximal and in spaced relation to a bottom surface of the cell culturecontainer, thereby defining within the cell culture container a lowervolume comprising the at least one tumor cell type and an upper volumecomprising a second surface of the porous membrane. The shear stress isapplied upon the second surface of the porous membrane in the uppervolume of the container.

In addition, in any of the methods wherein the at least one tumor celltype is plated on a first surface of a porous membrane, the method canfurther comprise depositing at least one extracellular matrix componenton the first surface of the porous membrane and plating the at least onetumor cell type on the at least one extracellular matrix component.Alternatively, the at least one tumor cell type can be suspended in asolution comprising at least one extracellular matrix component tocreate a suspension comprising the at least one tumor cell type and theat least one extracellular matrix component, and the suspension can bedeposited on the first surface of the porous membrane. The porousmembrane is suspended in the cell culture container such that the firstsurface is proximal and in spaced relation to a bottom surface of thecell culture container, thereby defining within the cell culturecontainer a lower volume comprising the at least one extracellularmatrix component and the at least one tumor cell type, and an uppervolume comprising a second surface of the porous membrane. The shearstress is applied upon the second surface of the porous membrane in theupper volume of the container.

The method can further comprise plating endothelial cells on the secondsurface of the porous membrane, and applying the shear stress upon theplated endothelial cells. The at least one tumor cell type is plated ona first surface of the porous membrane.

The method can further comprise plating at least one stromal cell typeon the second surface of the porous membrane and applying the shearstress upon the plated stromal cell type.

In methods where the at least one stromal cell type is plated on thesecond surface of the porous membrane, the method can further compriseplating endothelial cells on the second surface of the porous membrane.The at least one stromal cell type can be mixed with the endothelialcells prior to plating, and the method can comprise applying the shearstress upon the plated mixture of the at least one stromal cell type andthe endothelial cells. Alternatively, the at least one stromal cell typeand the endothelial cells can be sequentially plated on the secondsurface of the porous membrane. For example, the method can compriseplating the at least one stromal cell type on the second surface of theporous membrane, subsequently plating the endothelial cells on theplated stromal cell type, and applying the shear stress on the platedendothelial cells. Alternatively, the method can comprise plating theendothelial cells on the second surface of the porous membrane,subsequently plating the at least one stromal cell type on the platedendothelial cells, and applying the shear stress on the plated stromalcell type.

In the methods wherein the at least one tumor cell type is plated on afirst surface of a porous membrane, the porous membrane can be a firstporous membrane and the method can comprise plating the at least onetumor cell type on a first surface of the first porous membrane. Atleast one stromal cell type is plated on a second surface of the firstporous membrane. A second porous membrane is placed on the platedstromal cell type such that a first surface of the second porousmembrane contacts the plated stromal cells. The shear force is appliedupon a second surface of the second porous membrane.

In the methods comprising depositing at least one extracellular matrixcomponent or a suspension comprising the at least one tumor cell typeand at least one extracellular matrix component on the first surface ofa porous membrane the porous membrane can be a first porous membrane andthe method can further comprise depositing the at least oneextracellular matrix component on the first surface of the first porousmembrane and plating the at least one tumor cell type on the at leastone extracellular matrix component. Alternatively, the method canfurther comprise depositing the suspension comprising the at least onetumor cell type and the at least one extracellular matrix component onthe first surface of the first porous membrane. Either method furthercomprises plating at least one stromal cell type on a second surface ofthe first porous membrane, placing a second porous membrane on theplated stromal cell type such that a first surface of the second porousmembrane contacts the plated stromal cells, and applying the shear forceupon a second surface of the second porous membrane.

In the methods wherein the porous membrane is a first porous membranedescribed above, the method can further comprise plating endothelialcells on the second surface of the second porous membrane and applyingthe shear force upon the plated endothelial cells.

In the methods that comprise plating at least one tumor cell type orstromal cell type on a first surface of a porous membrane, the porousmembrane can be a first porous membrane and the method can furthercomprise plating the at least one stromal cell type on a first surfaceof a first porous membrane. A second porous membrane is placed on theplated stromal cell type, such that a first surface of the second porousmembrane contacts the plated stromal cells. At least one tumor cell typeis plated on a second surface of the second porous membrane. The shearforce is indirectly applied upon the at least one tumor cell type byapplying the shear stress upon the second surface of the first porousmembrane.

The present invention is also directed to another method for mimicking atumor microenvironment in vitro. The method comprises adding a culturemedium to a cell culture container and plating at least one stromal celltype on a first surface of a first porous membrane within the cellculture container. A second porous membrane is placed on the platedstromal cell type, such that a first surface of the second porousmembrane contacts the plated stromal cells. At least one tumor cell typeis plated on a second surface of the second porous membrane. Shearstress is indirectly applied upon the at least one tumor cell type byapplying a shear stress upon the second surface of the first porousmembrane, the shear stress resulting from flow of the culture mediuminduced by a flow device, the flow mimicking flow to which the tumorcells are indirectly exposed in vivo in the tumor microenvironment.

In the methods comprising plating at least one stromal cell type on afirst surface of a first porous membrane, the first porous membrane canbe suspended in the cell culture container such that the first surfaceof the first porous membrane is proximal and in spaced relation to abottom surface of the cell culture container, thereby defining withinthe cell culture container a lower volume comprising the at least onetumor cell type, the second porous membrane, and the at least onestromal cell type, and an upper volume comprising a second surface ofthe first porous membrane. The shear stress is applied upon the secondsurface of the first porous membrane in the upper volume of thecontainer.

In the methods comprising plating at least one stromal cell type on afirst surface of a first porous membrane, the method can furthercomprise depositing at least one extracellular matrix component on thesecond surface of the second porous membrane and plating the at leastone tumor cell type on the at least one extracellular matrix component.Alternatively, the method can further comprise suspending the at leastone tumor cell type in a solution comprising at least one extracellularmatrix component to create a suspension comprising the at least onetumor cell type and the at least one extracellular matrix component, anddepositing the suspension on the second surface of the second porousmembrane.

In the methods comprising plating at least one stromal cell type on afirst surface of a first porous membrane, the method can furthercomprise plating endothelial cells on the second surface of the firstporous membrane and applying the shear stress upon the platedendothelial cells.

In the methods comprising the use of a second porous membrane, themethod can further comprise immersing the second porous membrane in asolution comprising at least one extracellular matrix component prior toplacing the second porous membrane on the plated stromal cell type.

In any of the methods comprising plating endothelial cells, the methodcan further comprises coplating at least one tumor cell type with theendothelial cells. The coplating can comprise mixing the at least onetumor cell type with the endothelial cells prior to plating.Alternatively, the coplating can comprise sequentially plating the atleast one tumor cell type and the endothelial cells. For example, thecoplating can comprise plating the at least one tumor cell type andsubsequently plating the endothelial cells. Alternatively, the coplatingcan comprise plating the endothelial cells and subsequently plating theat least one tumor cell type.

In any of the methods comprising coplating at least one tumor cell typewith the endothelial cells, the coplating can comprise plating theendothelial cells and the at least one tumor cell type at a ratio ofabout 100:1 to about 3:1. For example, the coplating can compriseplating the endothelial cells and the at least one tumor cell type at aratio of about 50:1 to about 10:1.

In any of the methods comprising coplating at least one tumor cell typewith the endothelial cells, the at least one tumor cell type can be anyof the tumor cell types described herein. In these methods, the at leastone tumor cell type preferably comprises cells derived from aglioblastoma.

In any of the methods wherein cells are plated on a porous membrane, theporous membrane, the first porous membrane, or the second porousmembrane can adapted to permit fluid communication of the culture mediumand physical interaction and communication between cells plated onopposing sides of the porous membrane.

The present invention also relates to an in vitro method of testing adrug or a compound for an effect on a tumor. The method comprisesmimicking the tumor microenvironment, adding a drug or a compound to theculture medium, and indirectly applying the shear stress upon the atleast one tumor cell type directly or indirectly exposed to the drug orthe compound. A change in the at least one tumor cell type, in thepresence of the drug or the compound, indicates that the drug or thecompound has an effect on the tumor. The tumor microenvironment can bemimicked by the in vitro methods of mimicking a tumor microenvironmentdescribed above.

To confirm that the in vivo tumor microenvironment is mimicked, a changein the level or localization of a marker of the tumor microenvironmentcan be compared between a method of the invention and the same method inthe absence of the application of the shear stress. The level orlocalization of the marker in the at least one tumor cell type, the atleast one stromal cell type, or the endothelial cells upon applicationof the shear stress is compared to the level or localization of themarker in the at least one tumor cell type, the at least one stromalcell type, or the endothelial cells in the absence of the application ofshear stress. Alternatively, the level of a marker in the culture mediumupon application of the shear stress is compared to the level of themarker in the culture medium in the absence of the application of theshear stress. For example, if a marker is known to be associated withthe presence of a tumor and its concentration is known to increase inthe serum when a tumor is present in vivo, an increase in the level ofthe marker in the culture medium of the method of the invention withapplication of the shear stress as compared to the level of the markerin the culture medium in the absence of the application of the shearstress confirms that the tumor microenvironment is mimicked by the invitro method of the invention.

In any of the above methods, the cell culture container can compriseinlets and outlets. The inlets and outlets can be used for perfusingcell culture medium, drugs, compounds, and other components into and outof the cell culture container.

The inlets and outlets can be within the portions of the cell culturecontainer defining the upper and lower volumes.

Any of the methods can comprise perfusing culture medium into and out ofthe cell culture container. For example, the methods can compriseperfusing culture medium into and out of the upper volume and/orperfusing culture medium into and out of the lower volume.

Any of the methods for mimicking a tumor microenvironment in vitro cancomprise mimicking the tumor microenvironment in vitro in a first cellculture container according to any of the methods described herein,mimicking the tumor microenvironment in vitro in a second cell culturecontainer according to any of the methods described herein, andtransferring cells of the at least one tumor cell type cultured in thefirst cell culture container into the second cell culture container. Thetransferring can comprise manually transferring the cells of the atleast one tumor cell type cultured in the first cell culture containerinto the second cell culture container. Alternatively, an outlet in thefirst cell culture container can be connected to an inlet in the secondcell culture container, and the method can further comprise pumpingculture medium comprising the at least one tumor cell type out of thefirst cell culture container and into the second cell culture container.

In any of the methods for mimicking a tumor microenvironment in vitrodescribed herein, the method can further comprise introducing cellscultured in an in vitro system that models an organ or tissue into thecell culture container.

Cell Types

Any of the methods described above can further comprise plating at leastone stromal cell type on the surface within the cell culture container,on the at least one extracellular matrix component, or on the firstsurface of the porous membrane. Alternatively, at least one stromal celltype can be suspended with the tumor cell type in the solutioncomprising the at least one extracellular matrix component to create asuspension comprising the at least one stromal cell type, the at leastone tumor cell type, and the at least one extracellular matrixcomponent, and the suspension can be deposited on the surface within thecell culture container or on the first surface of the porous membrane.

For methods wherein the at least one tumor cell type is plated on afirst surface of a first porous membrane, the methods can furthercomprise plating at least one stromal cell type on the first surface ofthe first porous membrane. Alternatively, the methods can furthercomprise suspending at least one stromal cell type with the tumor celltype in the solution comprising the at least one extracellular matrixcomponent to create a suspension comprising the at least one stromalcell type, the at least one tumor cell type, and the at least oneextracellular matrix component, and depositing the suspension on thefirst surface of the first porous membrane.

For methods wherein the at least one tumor cell type is plated on asecond surface of a second porous membrane, the methods can furthercomprise plating at least one stromal cell type on the second surface ofthe second porous membrane. Alternatively, the methods can furthercomprise suspending at least one stromal cell type with the tumor celltype in the solution comprising the at least one extracellular matrixcomponent to create a suspension comprising the at least one stromalcell type, the at least one tumor cell type, and the at least oneextracellular matrix component, and depositing the suspension on thesecond surface of the second porous membrane.

Any of the methods described above can also further comprise plating oneor more additional cell types on a surface of the cell culturecontainer, on the at least one extracellular matrix component, on thefirst or second surface of the porous membrane, on the first or secondsurface of the first porous membrane, or on the first or second surfaceof the second porous membrane; or suspending one or more additional celltypes in the culture medium within the upper volume or in the culturemedium within the lower volume.

In any of the methods that comprise suspending the at least one tumorcell type in a solution comprising at least one extracellular matrixcomponent, the method can further comprise suspending one or moreadditional cell types with the at least one tumor cell type in thesolution comprising the at least on extracellular matrix component tocreate a suspension comprising the one or more additional cell types,the at least one tumor cell type, and the at least one extracellularmatrix component, and depositing the suspension on the surface withinthe cell culture container, on the first surface of the porous membrane,on the first surface of the first porous membrane, or on the secondsurface of the second porous membrane.

Cell types for use in the methods of the invention include primary cellsand immortalized cells. The at least one tumor cell type, theendothelial cells, the at least one stromal cell type, or the one ormore additional cell types can comprise immortalized cells. The at leastone tumor cell type, the endothelial cells, the at least one stromalcell type, or the one or more additional cell types can comprise primarycells.

Any of the cell types can comprise cells derived from an animal, e.g.,from a genetically modified animal or a human.

In any of the methods of the invention, the method can further comprisethe step of culturing the cell type or cell types.

The at least one tumor cell type, the endothelial cells, the at leastone stromal cell type, and additional cell types that can be used in themethods are further described below.

Tumor Cells

The at least one tumor cell type can comprise cells derived from acarcinoma, a sarcoma, a lymphoma, an adenosquamous carcinoma, a mixedmesodermal tumor, carcinosarcoma, a teratocarcinoma, or a combinationthereof.

The cells derived from a carcinoma can comprise cells derived from anadenocarcinoma, cells derived from a squamous cell carcinoma, or acombination thereof.

The cells derived from a sarcoma can comprise cells derived from anosteosarcoma, a chondrosarcoma, a leiomyosarcoma, a rhabdomyosarcoma, amesothelial sarcoma (mesothelioma), a fibrosarcoma, an angiosarcoma(e.g., from a hemangioendothelioma, a lymphangiosarcoma, or acombination thereof), a liposarcoma, a glioma, an astrocytoma, amyxosarcoma, a mesenchymous tumor, a mixed mesodermal tumor, or acombination thereof.

The cells derived from a lymphoma can comprise cells derived from aHodgkin lymphoma, a non-Hodgkin lymphoma, or a combination thereof.

The at least one tumor cell type can be derived from a tumor ofconnective tissue, a tumor of endothelium or mesothelium, a tumor oflymphoid tissue, a tumor of muscle, a tumor of an epithelial tissue, atumor of a neural tissue, a tumor of the amine precursor uptake anddecarboxylation (APUD) system, a tumor of a neural crest-derived cell, agonadal tumor, or a combination thereof.

Where the at least one tumor cell type is derived from a tumor ofconnective tissue, the tumor can comprise a tumor of adult fibroustissue (e.g., a firboma or a fibrosarcoma), embryonic (myxomatous)fibrous tissue (e.g., a myxoma or a myxosarcoma), adipose tissue (e.g.,a lipoma or a liposarcoma), cartilage tissue (e.g., a chondroma or achondrosarcoma), bone (e.g., osteoma or a osteosarcoma), notochord(e.g., a chordoma), a fibrous histiocytoma (e.g., a malignant fibroushistiocytoma), or a combination thereof.

Where the at least one tumor cell type is derived from a tumor ofendothelium or mesothelium, the tumor can comprise a blood vessel tumor(e.g., a hemangioma, a hemangiopericytoma, a hemangiosarcoma, or anangiosarcoma), a lymph vessel tumor (e.g., a lymphangioma or alymphangiosarcoma), a mesothelium tumor (e.g., a mesothelioma), or acombination thereof.

Where the at least one tumor cell type is derived from a tumor oflymphoid tissue, the tumor can comprises a plasmacytoma, a Hodgkinlymphoma, a non-Hodgkin lymphoma, or a combination thereof.

Where the at least one tumor cell type is derived from a tumor ofmuscle, the tumor can comprise a smooth muscle tumor (e.g., a leiomyomaor a leiomyosarcoma), a striated muscle tumor (e.g., a rhabdomyoma or arhabdomyosarcoma), or a combination thereof.

Where the at least one tumor cell type is derived from a tumor of anepithelial tissue, the tumor can comprise a tumor of a stratifiedsquamous tissue (e.g., a papilloma, a seborrheic keratosis, a skinadnexal tumor, a squamous cell carcinoma, or an epidermoid carcinoma), atumor of a glandular epithelium (e.g., a tumor of the glandularepithelium or a liver, kidney or bile duct), a tumor of transitionalepithelium (e.g., a transitional cell papilloma or a transitional cellcarcinoma), a placental tumor (e.g., a hydatidiform mole or achoriocarcinoma), a testicular tumor (e.g., a seminoma or an embryonalcell carcinoma), or a combination thereof. Where the tumor of theglandular epithelium is a tumor of the glandular epithelium of theliver, the tumor can comprise a hepatic adenoma or a hepatocellularcarcinoma. Where the tumor of the glandular epithelium is a tumor of theglandular epithelium of the kidney, the tumor can comprise a renaltubular adenoma, a renal cell carcinoma, or a hypernephroma. Where thetumor of the glandular epithelium is a tumor of the glandular epitheliumof the bile duct, the tumor can comprise a bile duct adenoma or acholangiocarcinoma.

Where the at least one tumor cell type is derived from a tumor of aneural tissue, the tumor can comprise a glial cell tumor (e.g., a gliomaor a glioblastoma), a nerve cell tumor (e.g., a ganglioneuroma, aneuroblastoma, or a medulloblastoma), a tumor of the meninges (e.g., ameningioma), a nerve sheath tumor (e.g., a Schwannoma, a neurilemmoma, aneurofibroma, a minigioma, or a neurofibrosarcoma), or a combinationthereof.

Where the at least one tumor cell type is derived from a tumor of theamine precursor uptake and decarboxylation (APUD) system, the tumor cancomprise a pituitary tumor (e.g., a basophilic adenoma, a eosinophilicadenoma, or a chromophobe adenoma), a parathyroid tumor (e.g., aparathyroid adenoma or a parathyroid carcinoma), a thyroid tumor (e.g.,a C cell hyperplasia or a medullary carcinoma of the thyroid), abronchial lining tumor (e.g., a bronchial carcinoid or an oat cellcarcinoma), an adrenal medulla tumor (e.g., a pheochromocytoma), apancreatic tumor (e.g., an islet celladenoma, an insulinoma, agastrinoma, or an islet cell carcinoma), a tumor of the stomach orintestines (e.g., a carcinoid), a tumor of the carotid body tumor orchemoreceptor system (e.g., a chemodectoma, a paraganglioma, or acarcinoid), or a combination thereof.

Where the at least one tumor cell type is derived from a tumor of aneural crest-derived cell, the tumor can comprise a tumor of a pigmentproducing cell (e.g., a nevus or a melanoma), a tumor of a Schwann cellof the peripheral nervous system (e.g., a Schwannoma or a neurilemmoma),a tumor of a Merkel cell (e.g., a Merkel cell neoplasm), or acombination thereof.

Where the at least one tumor cell type is derived from a gonadal tumor,the gonadal tumor can comprises a tumor of the ovary, a tumor of thetestis, a seminoma, a dysgerminoma, a choriocarcinoma, an embryonalcarcinoma, an endodermal sinus tumor, a teratocarcinoma, aSertoli-Leydig cell tumor, an arrhenoblastoma, a granulosa-theca celltumor, a hilar cell tumor, a lipid cell tumor, or a combination thereof.

The at least one tumor cell type can be derived from a tumor of thelung, breast, colon, rectum, prostate, bladder, bone, pancreas, liver,bile duct, ovary, testis, uterus, placenta, brain, cartilage, smoothmuscle, striated muscle, membranous lining of a body cavity, fibroustissue, blood vessel, lymph vessel, lymph node, adipose tissue,neurogenic connective tissue of the brain, kidney, pituitary gland,parathyroid, thyroid, bronchial lining, adrenalmedulla, stomach, largeintestine, small intestine, carotid body, chemoreceptor system, skin,gall bladder, or a combination thereof.

The at least one tumor cell type can comprise immortalized cells. Forexample, the at least one tumor cell type can comprise an immortalizedcell line comprising non-small cell lung adenocarcinoma cells, breastcarcinoma cells, pancreas carcinoma cells, prostate cancer cells,ovarian carcinoma cells, colon cancer cells, or a combination thereof.For example, the immortalized cell line can comprise human non-smallcell lung adenocarcinoma cell line A549, human breast carcinoma cellline MDA-MB-231, human pancreas carcinoma cell line BxPC-3, humanprostate cancer cell line DU145, human prostate cancer cell line LNCaP,human ovarian carcinoma cell line SKOV-3, human colon cancer cell lineCOLO-205, or a combination thereof.

The at least one tumor cell type can comprise primary cells. Forexample, the tumor cell type can comprise primary tumor cells obtainedfrom a subject by biopsy, tumor resection, blood draw, or a combinationthereof. A blood draw can be used to obtain cancer cells that have beenshed from the primary tumor and that are present in the circulatorysystem. The primary tumor cells can be obtained from a stage I tumor, astage II tumor, a stage III tumor, or a stage IV tumor.

The at least one tumor cell type can comprise tumor cells derived from ahumanized animal bearing a tumor derived from a human subject, such as ahumanized mouse. For example, the humanized mouse can be a non-obesediabetic severe combined immunodeficiency (NOD SCID) mouse, aNOD/Shi-scid/IL-2Rγnull (NOG) mouse, or a NOD SCID IL-2Rγ knockout (NSG)mouse.

Endothelial Cells

The endothelial cells can comprise microvascular endothelial cells,macrovascular endothelial cells, endothelial progenitor cells, or acombination thereof.

The endothelial cells can be derived from a tumor. For example, wherethe at least one tumor cell type comprises cells derived from a tumor ofan animal, the endothelial cells can be derived from the same tumor.

The endothelial cells can also be derived from an organ or tissue inwhich a tumor resides. For example, where the at least one tumor celltype comprises cells derived from a tumor of an animal, the endothelialcells can be derived from the organ or tissue in which that tumorresides. Thus, for instance, if the at least one tumor cell typecomprises cells derived from a tumor of the lung, the endothelial cellscan comprise endothelial cells derived from lung tissue of that animalor lung tissue of a different animal.

The endothelial cells can comprise endothelial cells derived from lung,breast, colon, rectum, prostate, bladder, bone, pancreas, liver, bileduct, ovary, testis, uterus, placenta, brain, cartilage, smooth muscle,striated muscle, a membranous lining of a body cavity, fibrous tissue,blood vessel, lymph vessel, lymph node, adipose tissue, neurogenicconnective tissue of the brain, kidney, pituitary gland, parathyroid,thyroid, bronchial lining, adrenalmedulla, stomach, large intestine,small intestine, carotid body, chemoreceptor system, skin, gall bladder,or a combination thereof.

For example, the endothelial cells can comprise lung microvascularendothelial cells, breast microvascular endothelial cells, pancreaticmicrovascular endothelial cells, prostate microvascular endothelialcells, ovarian microvascular endothelial cells, colon microvascularendothelial cells, or a combination thereof.

The endothelial cells can comprise cells derived from induciblepluripotent stem cells (iPSC).

Stromal Cells

The at least one stromal cell type can comprise fibroblasts, immunecells, pericytes, inflammatory cells, or a combination thereof.

Where the at least one stromal cell type comprises fibroblasts, thefibroblasts can comprise fetal stromal fibroblasts, for example, humanfetal stromal fibroblast cell line IMR-90. Alternatively, thefibroblasts can comprise human lung fibroblast cell line Hs888Lu.

Where the at least one stromal cell type comprises immune cells, theimmune cells can comprise macrophages, lymphocytes, dendritic cells, ora combination thereof.

Where the at least one stromal cell type comprises inflammatory cells,the inflammatory cells can comprise B cells, T cells, or a combinationthereof.

The at least one stromal cell type can comprise cells derived frominducible pluripotent stem cells (iPSC).

The at least one stromal cell type can be mixed with the at least onetumor cell type prior to plating. For example, the at least one stromalcell type can be mixed with the at least one tumor cell type at a ratioof about 0.1:1 to about 3:1, a ratio of about 0.2:1 to about 2:1, aratio of about 0.25:1, or a ratio of about 1:1.

Alternatively, the method can comprise sequentially plating the at leastone tumor cell type and the at least one stromal cell type. For example,the method can comprise plating the at least one tumor cell type andsubsequently plating the at least one stromal cell type on the platedtumor cell type. Alternatively, the method can comprise plating the atleast one stromal cell type and subsequently plating the at least onetumor cell type on the plated stromal cell type.

Additional Cell Types

The methods described herein can also further comprise plating one ormore additional cell types on a surface of the cell culture container,on the at least one extracellular matrix component, on the first orsecond surface of the porous membrane, on the first or second surface ofthe first porous membrane, or on the first or second surface of thesecond porous membrane; or suspending one or more additional cell typesin the culture medium within the upper volume or in the culture mediumwithin the lower volume. For example, the one or more additional celltypes can comprise a cell type adhered to the bottom surface of the cellculture container.

In any of the methods that comprise suspending the at least one tumorcell type in a solution comprising at least one extracellular matrixcomponent, the method can further comprise suspending one or moreadditional cell types with the at least one tumor cell type in thesolution comprising the at least on extracellular matrix component tocreate a suspension comprising the one or more additional cell types,the at least one tumor cell type, and the at least one extracellularmatrix component, and depositing the suspension on the surface withinthe cell culture container, on the first surface of the porous membrane,on the first surface of the first porous membrane, or on the secondsurface of the second porous membrane.

The one or more additional cell types can comprise fibroblasts, immunecells, pericytes, inflammatory cells, or a combination thereof.

Where the one or more additional cell types comprise fibroblasts, thefibroblasts can comprise fetal stromal fibroblasts, for example, humanfetal stromal fibroblast cell line IMR-90. Alternatively, thefibroblasts can comprise human lung fibroblast cell line Hs888Lu.

Where the one or more additional cell types comprises immune cells theimmune cells can comprise macrophages, lymphocytes, dendritic cells, ora combination thereof. For example, the immune cells can comprise thelymphocytes and the lymphocytes can be suspended in the culture mediumwithin the upper volume.

Where the one or more additional cell types comprises inflammatorycells, the inflammatory cells can comprise B cells, T cells, or acombination thereof.

Extracellular Matrix Components

The one or more extracellular matrix components can be produced by acell type plated on a surface within the cell culture container (e.g.,by the at least one tumor cell type). When the extracellular matrix isproduced by a cell type or cell types plated on a surface within thecell culture container, the extracellular matrix is referred to hereinas “endogenous” extracellular matrix.

By contrast, when one or more extracellular matrix components aredeposited on a surface within the cell culture container during themethods described herein, the extracellular matrix is referred to as“exogenous” or “exogenously added” extracellular matrix.

The methods described herein can comprise culturing the cell type orcell types in the substantial absence of exogenously added extracellularmatrix. The “substantial absence of exogenously added extracellularmatrix” means that the method does not comprise depositing anextracellular matrix component on a surface within the cell culturecontainer, or suspending one or more cell types in a solution comprisingan extracellular matrix component to create a suspension comprising thecell type and the at least one extracellular matrix component anddepositing the suspension on a surface within the cell culturecontainer. However, where the methods comprise the use of first andsecond porous membranes, the method can comprise immersing the secondporous membrane in a solution comprising at least one extracellularmatrix component prior to placing the second porous membrane on theplated stromal cell type. Without being bound to any particular theory,it is thought that when this immersion step is performed, theextracellular matrix component is absorbed by the porous membrane andaids in the attachment of cells to the membrane, but only results in theaddition of a very small amount of extracellular matrix to the cellculture container. Thus, cells can be cultured in the “substantialabsence of exogenously added extracellular matrix” even where thisimmersion step has been performed. Culturing cells in the “substantialabsence of exogenously added extracellular matrix” also includes methodsthat do not comprise adding any exogenous extracellular matrixwhatsoever to the cell culture container.

Extracellular matrix components for use in the methods of the inventioncan comprise a collagen, heparan sulfate, chondroitin sulfate, keratansulfate, hyaluronic acid, an elastin, a fibronectin, a laminin, avitronectin, or a combination thereof. The extracellular matrixcomponent is preferably a type of extracellular matrix component that ispresent in the in vivo environment of the tumor cells, endothelialcells, stromal cells, and/or one or more additional cell types.

For example, where the extracellular matrix component comprises acollagen, the collagen can comprise collagen type I, collagen type II,collagen type III, collagen type IV, collagen type V, collagen type VI,collagen type VII, collagen type VIII, collagen type IX, collagen typeX, collagen type XI, collagen type XII, collagen type XIII, collagentype XIV, collagen type XV, collagen type XVI, collagen type XVII,collagen type XVIII, collagen type XIX, collagen type XX, collagen typeXXI, collagen type XXII, collagen type XXIII, collagen type XXIV,collagen type XXV, collagen type XXVI, collagen type XXVII, collagentype XXVIII, or a combination thereof. Where the extracellular matrixcomponent comprises a collagen, the concentration of the collagen ispreferably about 1 mg/ml to about 10 mg/ml, about 2 mg/ml to about 5mg/ml, or at least about 2 mg/ml.

The extracellular matrix component can comprise decellularizedextracellular matrix purified from a biological source (e.g., humanplacenta).

The extracellular matrix component can be secreted by a cell type orcell types within the cell culture container (e.g., by the at least onetumor cell type).

The extracellular matrix component can be secreted by fibroblasts,chondrocytes, or osteoblasts plated on a surface within the cell culturecontainer.

The extracellular matrix component suitably mimics the stiffness of thein vivo tumor microenvironment. For example, the at least oneextracellular matrix component can have a Young's modulus of about 0.1kPa to about 25 kPa, about 0.15 kPa to about 15 kPa, or about 3 kPa toabout 12 kPa.

The at least one extracellular matrix component can have a non-uniformYoung's modulus. For example, two or more different types of anextracellular matrix component or two or more concentrations of a singleextracellular matrix component can be deposited on the surface withinthe cell culture container or the surface of the porous membrane tocreate a layer of extracellular matrix that has a non-uniform Young'smodulus. The Young's modulus of the extracellular matrix can vary in alinear gradient across the surface within the cell culture container oracross the surface of the porous membrane. Alternatively the Young'smodulus of the extracellular matrix can vary in a concentric gradient onthe surface within the cell culture container or the surface of theporous membrane.

The methods of the invention can also comprise depositing an additionallayer of at least one extracellular matrix component on top of the atleast one tumor cell type, such that the at least one extracellularmatrix component substantially surrounds the at least one tumor celltype. The additional layer of at least one extracellular matrixcomponent can have a Young's modulus that is different from the Young'smodulus of the at least one extracellular matrix component deposited onthe surface within the cell culture container, on the first surface ofthe porous membrane, on the first surface of the first porous membrane,or on the second surface of the second porous membrane. Alternatively,the additional layer of at least one extracellular matrix component canhave a Young's modulus that is substantially the same as the Young'smodulus of the at least one extracellular matrix component deposited onthe surface within the cell culture container, on the first surface ofthe porous membrane, on the first surface of the first porous membrane,or on the second surface of the second porous membrane. The Young'smodulus of the additional layer of at least one extracellular matrixcomponent can also be non-uniform.

In any of the methods that comprise the addition of one or moreexogenous extracellular matrix components, the method can comprise theaddition of single or multiple layers of ECM.

In any of the methods that comprise the addition of one or moreexogenous extracellular matrix components, the exogenous extracellularmatrix can comprise a single ECM protein or a mixture of multiple ECMproteins. For example, the exogenous extracellular matrix can comprise amixture of collagen, fibronectin, and/or laminin.

Cell Culture Medium

Standard culture medium can be used in the methods of the invention. Thecomposition of the culture medium will vary depending on the particularcell type(s) being cultured.

Additional components can also be included in the culture medium. Forexample, factors that are known to influence adipogenesis, such asGM-CSF and TGF-β, can be added to the culture medium. In vivo, thesefactors are secreted by macrophages.

The culture medium can comprise sera, blood, blood cells, a bloodcomponent, immune cells, conditioned culture medium, or a combinationthereof.

The sera, blood, blood cells, blood component, or immune cells can bederived from a human or an animal (e.g., a mouse, rat, guinea pig,hamster, rabbit, cat, dog, monkey, cow, pig, horse, goat, sheep, bird,or fish).

The immune cells can comprise B cells, dendritic cells, granulocytes,innate lymphoid cells, megakaryocytes, monocytes, macrophages, naturalkiller cells, T cells, thymocytes, or a combination thereof.

The blood cells can comprise platelets, red blood cells, or acombination thereof.

The blood component can comprises a clotting factor, a lipoprotein, atriglyceride, or a combination thereof.

The conditioned culture medium can comprises conditioned culture mediumfrom a culture comprising tumor cells, a culture comprising endothelialcells, a culture comprising a stromal cell type, or a combinationthereof.

Flow Devices

The shear stress can be applied using any suitable flow device which iscapable of inducing flow of the culture media, wherein the flow mimicsflow to which the cell type or cell types being cultured are exposed invivo in the tumor microenvironment. For example, the flow device can bea cone-and-plate device or a parallel plate flow device.

The flow device can be a cone-and-plate device substantially asdescribed in U.S. Pat. No. 7,811,782 and in Hastings, et al.,Atherosclerosis prone hemodynamics differentially regulates endothelialand smooth muscle cell phenotypes and promotes pro-inflammatory priming,AMERICAN J. PHYSIOLOGY & CELL PHYSIOLOGY 293:1824-33 (2007), thecontents of each of which are hereby incorporated by reference withrespect to their teachings regarding cone-and-plate flow devices. Anexample of such a device is depicted in FIG. 1. The device 200 comprisesan electronic controller for receiving a set of electronic instructions,a motor 220 operated by the electronic controller, and a shear stressapplicator operatively connected to the motor for being driven by themotor. The shear stress applicator can comprise a cone 7, which isattached to the motor, and the cone can be directly driven by the motor.The motor causes the cone to rotate in either direction (clockwise orcounterclockwise). The device further comprises a Z-axis micrometer 210that allows the cone 7 of the device to be raised and lowered.

The cone-and-plate device accommodates a cell culture container 1, forexample a Petri dish (e.g., a 75-mm diameter Petri dish). The cone 7 canbe adapted to fit inside the cell culture container. Thus, for example,in a device adapted for use with 75-mm diameter Petri dishes, the conehas a diameter of about 71.4 mm. The cone generally has a shallow coneangle. For example, the angle between the surface of the cone and thesurface within the Petri dish is approximately 0.5°-2° (e.g., 1°).

When the cone 7 of the device 200 is submerged in culture media 3 in thecell culture container 1 and rotated by the motor 220, the cone exerts arotational stress upon the culture media, and this in turn applies shearstress to cells plated within the cell culture container or to a surfaceof a porous membrane 11 suspended in the cell culture container. Forexample, cells 6 can be plated on a first surface of a porous membrane,the cone can be used to apply a shear stress to the opposing surface ofthe membrane as depicted in the inset in FIG. 1.

A porous membrane can be suspended in the cell culture container using acell culture insert 4 that includes a porous membrane 11 and a support10. The cell culture insert is adapted to fit inside the cell culturecontainer. The cell culture insert suitably has a height that is shorterthan the height of the cell culture container, such that when the cellculture insert is placed into the cell culture container, the supportportion of the cell culture insert contacts the perimeter of the cellculture container and holds the porous membrane in a suspended positionwithin the cell culture container. For example, the insert can have arim that extends around the perimeter of the insert, wherein the rim ofthe insert then contacts the perimeter of the cell culture container tosuspend the insert in the cell culture container. Cell culture insertsin a variety of sizes are commercially available from a number ofmanufacturers (e.g., Corning, which manufactures TRANSWELL inserts;Millicell; and ThinCert). The porous membranes can be made of anysuitable porous material (e.g., polyester, polycarbonate,collagen-coated polytetrafluoroethylene (PTFE), or polyethyleneterephthalate (PET)) and can have a variety of thicknesses and poresizes.

The cone-and-plate device can also include a base 240 for securelyholding the cell culture container. The device can also include clipsthat mount on the cell culture container dish and secure inflow andoutflow tubing, which is used to perfuse the upper and lower volumes, asdescribed further below.

The flow can be derived from a previously measured hemodynamic pattern,and can be modeled into a set of electronic instructions. The shearstress is based on the set of electronic instructions.

The flow device can comprise a body adapted for being positioned in theculture medium in the upper volume of the cell culture container and amotor adapted to rotate the body. The body can have a conical surface ora flat surface.

The flow device can be adapted for positioning the conical or flatsurface of the body in the cell culture container and in contact withthe culture medium.

The flow device can comprise an electronic controller for receiving theset of electronic instructions. The motor is operated by the electroniccontroller. A shear stress applicator operatively connected to the motoris driven by the motor. Preferably, the shear stress applicatorcomprises a cone or a disc attached to the motor.

The flow device is used in conjunction with a cell culture container.The cell culture container can include inlets and outlets for perfusingcell culture medium, drugs, compounds, and other components into and outof the cell culture container.

The inlets and outlets can be secured to the cell culture container byclips. FIG. 2 depicts a clip. Each clip 300 is made up of three parts:the main body 301 and two pieces of thin metal tubing 302 and 303 asshown in FIG. 2. The clip 300 can be secured to the side of a cellculture container from the outside by a screw 304. For example, twoclips can be attached and tightened to the side of the container fromthe outside by a screw 304. The main body 301 is made of treatedstainless steel metal and angles around the edge of the dish forattachment and access purposes. Two pieces of thin metal tubing (302 and303) per clip are bent to provide access to the dish for supplying anddrawing off media efficiently, without obstructing the cone rotation. Aset screw 305 on either side of the main body 301 secures the metaltubing 302, 303 to the main body and holds the metal tubing in placesuch that it extends to the correct depth within the culture media.Flexible tubing then slides over the metal tubing, which is used toperfuse media into and out of the cell culture container (e.g., from asource bottle to the container via mechanical peristaltic pump).

FIG. 3 shows two clips 300 positioned in a cell culture container 1. Inthe configuration shown in FIG. 3, a porous membrane 11 is suspended inthe cell culture container. FIG. 3 also depicts the cone 7 of acone-and-plate flow device and the culture medium 3.

As the cone of the cone-and-plate device rotates, fluid is transportedin a concentric manner within the upper volume of the cell culturecontainer. In addition, the rotation of the cone causes a downward flowof cell culture medium through the porous membrane and through any cellsplated on the porous membrane and/or extracellular matrix componentsdeposited on the porous membrane.

Hemodynamic Patterns

The flow can be derived from a previously measured hemodynamic pattern.

The hemodynamic pattern can be derived from the vasculature of a tumor.

The hemodynamic pattern can be derived from at least a portion of acapillary, an arteriole, an artery, a venule, or a vein.

The hemodynamic pattern can be derived from at least a portion of anorgan. For example, the hemodynamic pattern can be derived from a liver,a kidney, a lung, a brain, a pancreas, a spleen, a large intestine, asmall intestine, a heart, a skeletal muscle, an eye, a tongue, areproductive organ, or an umbilical cord.

The hemodynamic pattern can be derived from analysis of ultrasound data.

The hemodynamic pattern can be derived from analysis of magneticresonance imaging (MRI) data.

The flow or the hemodynamic pattern can be time-variant.

The flow or the hemodynamic pattern can be derived from an animal, suchas a genetically modified animal or a human. Preferably, the hemodynamicpattern is derived from a human.

For example, FIG. 4A shows a Doppler sonography image of the centralbronchial artery in a patient diagnosed with a pulmonary lesion. Themono-phasic low-impedance flow signal is indicative of a malignantlesion. FIG. 4B depicts wall shear stress calculations (dynes/cm²) ofthe Doppler flow signal of a human pulmonary lesion. FIG. 4C provides aschematic illustration of an exemplary arterial blood supply in apulmonary lesion.

The shear stress applied upon the at least one tumor cell type can beabout 0.1 dynes/cm² to about 200 dynes/cm². For example, the shearstress applied upon the at least one tumor cell type can be about 0.1dynes/cm² to about 100 dynes/cm².

The shear stress can be applied at a rate of about 1 sec⁻¹ to about 1000sec⁻¹.

Exemplary Methods for Mimicking a Tumor Microenvironment In Vitro

FIGS. 5 through 10 are schematic diagrams illustrating exemplary methodsfor mimicking a tumor microenvironment in vitro. In each of FIGS. 5-10,a cell culture container 1 contains a culture medium 3.

In FIGS. 5-8, the cell culture container also contains a porous membrane11. The porous membrane is suspended in the cell culture container suchthat a first surface of the porous membrane is proximal and in spacedrelation to the bottom surface of the cell culture container, therebydefining within the cell culture container a lower volume 23 comprisingthe tumor cells 5 and an upper volume 25 comprising a second surface ofthe porous membrane. The porous membrane shown in FIGS. 5-8 can be aporous membrane of a cell culture insert adapted to fit inside the cellculture container.

In FIGS. 5-8, at least one extracellular matrix component (ECM) 9 ispresent on the first surface of the porous membrane. The extracellularmatrix component can be endogenously produced by cells plated within thecell culture container. Alternatively, exogenous extracellular matrixcan be deposited on the first surface of the porous membrane by any ofthe methods described herein. The extracellular matrix can include bothendogenous extracellular matrix produced by cells plated within the cellculture container and exogenously added extracellular matrix.

In FIGS. 5-8, tumor cells 5 are also present on the first surface of theporous membrane and are substantially surrounded by the ECM. In FIGS. 6and 7, both tumor cells 5 and stromal fibroblasts 15 are also present onthe first surface of the porous membrane and are substantiallysurrounded by the ECM. In FIG. 6, the stromal fibroblasts and tumorcells are plated sequentially, with the stromal fibroblasts being platedfirst, and the tumor cells being plated on the plated stromalfibroblasts. In FIG. 7, the stromal fibroblasts and tumor cells aremixed together with one another prior to plating.

In each of FIGS. 5-8, endothelial cells 13 are plated on the secondsurface of the porous membrane. In FIG. 8, stromal fibroblasts 15 arealso plated on the second surface of the porous membrane. In FIG. 8, thestromal fibroblasts and endothelial cells are plated sequentially, withthe stromal fibroblasts being plated first on the second surface of theporous membrane, and the endothelial cells being plated on the platedstromal fibroblasts. Alternatively, although not depicted, the stromalfibroblasts and endothelial cells can be mixed together with one anotherprior to plating.

FIGS. 9 and 10 depict methods that use two porous membranes, a firstporous membrane and a second porous membrane. In FIG. 9, stromalfibroblasts 15 are plated on a first surface of a first porous membrane11. The porous membrane is suspended in the cell culture container suchthat a first surface of the first porous membrane is proximal and inspaced relation to the bottom surface of the cell culture container,thereby defining within the cell culture container a lower volume 23comprising the tumor cells 5 and an upper volume 25 comprising a secondsurface of the porous membrane. The first porous membrane 11 can be aporous membrane of a cell culture insert adapted to fit inside the cellculture container. A second porous membrane 12 is placed on the platedstromal cell type, such that a first surface of the second porousmembrane contacts the plated stromal fibroblasts. ECM 9 is present onthe second surface of the second porous membrane. The ECM can beendogenously produced by cells plated within the cell culture container.Alternatively, ECM can be deposited on the second surface of the secondporous membrane by any of the methods described herein. The ECM caninclude both endogenous ECM produced by cells plated within the cellculture container and exogenously added ECM. Tumor cells 5 are alsopresent on the second surface of the second porous membrane and aresubstantially surrounded by the ECM. Endothelial cells 13 are plated onthe second surface of the first porous membrane.

In FIG. 10, ECM 9 is present on the first surface of a first porousmembrane 11. The porous membrane is suspended in the cell culturecontainer such that a first surface of the porous membrane is proximaland in spaced relation to the bottom surface of the cell culturecontainer, thereby defining within the cell culture container a lowervolume 23 comprising the tumor cells 5 and an upper volume 25 comprisinga second surface of the porous membrane. The first porous membrane canbe a porous membrane of a cell culture insert adapted to fit inside thecell culture container. The ECM can be endogenously produced by cellsplated within the cell culture container. Alternatively, ECM can bedeposited on the first surface of the first porous membrane by any ofthe methods described herein. The ECM can include both endogenous ECMproduced by cells plated within the cell culture container andexogenously added ECM. Tumor cells 5 are also present on the firstsurface of the first porous membrane and are substantially surrounded bythe ECM. Stromal fibroblasts 15 are plated on a second surface of thefirst porous membrane. A second porous membrane 12 is placed on theplated stromal fibroblasts such that a first surface of the secondporous membrane contacts the plated stromal fibroblasts. Endothelialcells 13 are plated on the second surface of the second porous membrane.

FIGS. 5-10 also show inlets 17 and outlets 19 that can be used forperfusing cell culture medium, drugs, compounds, and other componentsinto and out of the cell culture container.

The cone 7 of a cone-and-plate flow device is also shown in FIGS. 5-10.The cone induces concentric flow of the culture medium within the uppervolume of the cell culture container, as represented by the dottedcircular arrow. The flow of the medium in turn applies a shear stressupon the endothelial cells. The dotted arrows pointing downwards towardsthe bottom of the cell culture container represent the downwardtransport of medium, drugs, and other components through the porousmembrane that occurs upon application of the shear stress and perfusionof culture medium into and out of the cell culture container.

Methods for Mimicking Tumor Metastasis

The present invention further relates to methods for mimicking tumormetastasis. The methods for mimicking tumor metastasis include methodsfor mimicking tumor metastasis in vitro, and methods for mimicking tumormetastasis in an animal.

Methods for Mimicking Tumor Metastasis In Vitro

A method for mimicking tumor metastasis in vitro is provided. The methodcomprises introducing cells of at least one tumor cell type culturedaccording to any one of the methods described above into an in vitrosystem that models an organ or tissue. For example, the in vitro systemthat models the organ or tissue can be an in vitro system that modelsthe liver, pancreas, bone, lung, blood vessels, the lymphatic system,brain, muscle, bladder, kidney, intestine, colon, gall bladder, skin, orbone.

In vitro systems that model the liver are described in U.S. PatentApplication Publication No. US 2013/0309677 and PCT Publication No.2013/0158939, the contents of both of which are hereby incorporated byreference in their entirety and which are described herein in thesection entitled “In vitro systems that model the liver,” Examples12-14, and FIGS. 20-43. An in vitro system that models the liverdescribed in U.S. Patent Application Publication No. US 2013/0309677comprises a cell culture container containing a culture medium and aporous membrane, wherein hepatocytes are plated on a first surface ofthe porous membrane and the porous membrane is suspended in the anothercell culture container such that the first surface is proximal and inspaced relation to a bottom surface of the container, thereby definingwithin the container a lower volume comprising the hepatocytes and anupper volume comprising a second surface of the porous membrane. A shearstress is applied upon the second surface of the porous membrane in theupper volume, the shear stress mimicking flow to which the hepatocytesare exposed in vivo. The cell culture container further comprises inletswithin the portions of the cell culture container defining the upper andlower volumes. The cell culture container can also comprise outletswithin the portions of the cell culture container defining the upper andlower volumes.

Thus, in the method for mimicking tumor metastasis in vitro, the invitro system that models the liver can comprise another cell culturecontainer comprising a culture medium and a porous membrane. Hepatocytesare plated on a first surface of the porous membrane, and the porousmembrane is suspended in the another cell culture container such thatthe first surface is proximal and in spaced relation to a bottom surfaceof the container, thereby defining within the container a lower volumecomprising the hepatocytes and an upper volume comprising a secondsurface of the porous membrane. A shear stress is applied upon thesecond surface of the porous membrane in the upper volume, the shearstress mimicking flow to which the hepatocytes are exposed in vivo. Theanother cell culture container further comprises inlets within theportions of the cell culture container defining the upper and lowervolumes.

In the in vitro system that models the liver described in U.S. PatentApplication Publication No. US 2013/0309677 and PCT Publication No.2013/0158939, at least one extracellular matrix component (e.g., acollagen) can be plated on the first surface of the porous membrane, andthe hepatocytes can be plated on the at least one extracellular matrixcomponent. An additional layer of at least one extracellular matrixcomponent can be deposited on top of the hepatocytes, such that the atleast one extracellular matrix component substantially surrounds thehepatocytes.

In the in vitro system that models the liver described in U.S. PatentApplication Publication No. US 2013/0309677 and PCT Publication No.2013/0158939, sinusoidal endothelial cells can be plated on the secondsurface of the porous membrane. Additional non-parenchymal cell types,such as hepatic stellate cells, Kupffer cells, or a combination thereof,can also be plated on the first or second surface of the porousmembrane.

FIG. 11 provides a schematic diagram illustrating an exemplary in vitrosystem that models the liver. A cell culture container 1′ contains aculture medium 3′. The cell culture container also contains a porousmembrane 11′. The porous membrane is suspended in the cell culturecontainer such that a first surface of the porous membrane is proximaland in spaced relation to the bottom surface of the cell culturecontainer, thereby defining within the cell culture container a lowervolume 23′ comprising hepatocytes and an upper volume 25′ comprising asecond surface of the porous membrane. The porous membrane can be aporous membrane of a cell culture insert adapted to fit inside the cellculture container. An extracellular matrix component (ECM) 9′ is presenton the first surface of the porous membrane. Hepatocytes 100 and Kupffercells 120 are also present on the first surface of the porous membraneand are substantially surrounded by the ECM. The ECM can be endogenouslyproduced by cells plated within the cell culture container.Alternatively, exogenous ECM can be deposited on the first surface ofthe porous membrane by any of the methods described herein. The ECM caninclude both endogenous ECM produced by cells plated within the cellculture container and exogenously added ECM. Sinusoidal endothelialcells 110 are plated on the second surface of the porous membrane.

FIG. 11 also shows inlets 17′ and outlets 19′ that can be used forperfusing culture medium, drugs, compounds, cells, and other componentsinto and out of the cell culture container.

FIG. 11 also depicts the cone 7′ of a cone-and-plate flow device. Thecone induces flow of the culture medium, as represented by the dottedcircular arrow in the upper volume. The flow of the medium in turnapplies a shear stress upon the endothelial cells. The dotted arrowspointing downwards towards the bottom of the cell culture containerrepresent the transport of medium, drugs, and other components thatoccurs upon application of the shear stress and perfusion of culturemedium into and out of the cell culture container.

In the method for mimicking tumor metastasis in vitro, the cells of theat least one tumor cell type can be introduced into the in vitro systemthat models the liver by transferring the cells of the at least onetumor cell type into the lower volume or the upper volume of the invitro system that models the liver. When the cells of the at least onetumor cell type are introduced into the upper volume of the in vitrosystem that models the liver, the method can further comprise assessingmigration of the cells of the at least one tumor cell type into thelower volume of the in vitro system that models the liver. Assessingmigration of the tumor cells from the upper volume to the lower volumecan be achieved by fixing the cells in the upper and lower chamber andperforming microscopic imaging of the cells in the upper and lowervolumes, and/or by sorting the cells in the upper or lower volumes wherethe tumor cells are labeled with a molecular tracer (e.g., aradiolabelled probe, a fluorescent protein, or colorometric tracer).

The transferring can comprise manually transferring the cells of the atleast one tumor cell type into the lower volume or the upper volume ofthe in vitro system that models the liver. For example, the cells of theleast one tumor cell type can be removed from the surface within thecell culture container or the porous membrane by trypsinization or byenzymatic digestion of the ECM, if present. The cells of the at leastone tumor cell type can then be transferred to the upper or lower volumeof the in vitro system that models the liver using a pipette.Alternatively, culture medium from the upper or lower volume of the cellculture container containing the at least one tumor cell type can bepipetted into the upper or lower volume of the in vitro system thatmodels the liver.

Alternatively, the transferring can comprise pumping cell culture mediumout of the upper or lower volume of the cell culture containercontaining the at least one tumor cell type and into the upper or lowervolume of the cell culture container of the in vitro system that modelsthe liver. This mimics the seeding of distal organs by tumor cells invitro. Such methods are illustrated in FIGS. 12A-D. In FIG. 12A, forexample, tubing 21 is used to connect to an outlet 19 within the lowervolume 23 of the cell culture container 1 comprising the at least onetumor cell type 5 to an inlet 17′ in the lower volume 23′ of the cellculture container 1′ of the in vitro system that models the liver. Theculture medium can be pumped through the tubing to transfer the cells ofthe at least one tumor cell type into the lower volume of the in vitrosystem that models the liver. The methods depicted in FIGS. 12B, C, andD are similar, but involve pumping culture medium from the lower volume23 of the cell culture container 1 that contains the at least one tumorcell type 5 into the upper volume 25′ of the cell culture container 1′of the in vitro system that models the liver (FIG. 12B), pumping culturemedium from the upper volume 25 of the cell culture container 1 thatcontains the at least one tumor cell type 5 into the lower volume 23′ ofthe cell culture container 1′ of the in vitro system that models theliver (FIG. 12C), or pumping culture medium from the upper volume 25 ofthe cell culture container 1 that contains the at least one tumor celltype 5 into the upper volume 25′ of the cell culture container 1′ of thein vitro system that models the liver (FIG. 12D).

Thus, the cell culture container comprising the at least one tumor celltype can further comprise an outlet within the portion of the cellculture container defining the lower volume and containing the at leastone tumor cell type. The outlet is connected to an inlet in the anothercell culture container of the in vitro system that models the liver. Thetransferring comprises pumping the culture medium out of the lowervolume of the cell culture container comprising the at least one tumorcell type and into the upper or lower volume of the another cell culturecontainer of the in vitro system that models the liver.

Alternatively, the cell culture container comprising the at least onetumor cell type can further comprise an outlet within the portion of thecell culture container defining the upper volume. The outlet isconnected to an inlet in the another cell culture container of the invitro system that models the liver. The transferring comprises pumpingthe culture medium out of the upper volume of the cell culture containerand into the upper or lower volume of the another cell culture containerof the in vitro system that models the liver.

The present invention further relates to another method for mimickingtumor metastasis in vitro. The method comprises adding a culture mediumto a cell culture container and plating at least one cell type on afirst surface of a porous membrane within the cell culture container,wherein the porous membrane is suspended in the cell culture containersuch that the first surface is proximal and in spaced relation to abottom surface of the cell culture container, thereby defining withinthe cell culture container a lower volume comprising the at least onecell type and an upper volume comprising a second surface of the porousmembrane. A shear stress is indirectly applied upon the at least onecell type, the shear stress resulting from flow of the culture mediuminduced by a flow device, the flow mimicking flow to which the cells areindirectly exposed in vivo. Tumor cells derived from a human or ahumanized animal are introduced into the upper volume or the lowervolume.

The at least one cell type can comprise hepatocytes or smooth musclecells.

The method can further comprise plating a second cell type on the secondsurface of the porous membrane. The second cell type can compriseendothelial cells.

Where the tumor cells are derived from a humanized animal, the humanizedanimal is suitably a humanized mouse, for example, a non-obese diabeticsevere combined immunodeficiency (NOD SCID) mouse, aNOD/Shi-scid/IL-2Rγnull (NOG) mouse, or a NOD SCID IL-2Rγ knockout (NSG)mouse.

The present invention further relates to an in vitro method of testing adrug or a compound for an effect on tumor metastasis. The methodcomprises mimicking tumor metastasis in vitro by any of the methodsdescribed above, and adding a drug or a compound to the culture medium.A change in the cells of the at least one tumor cell type in the invitro system that models the organ or tissue, in the presence of thedrug or the compound, indicates that the drug or the compound has aneffect on tumor metastasis.

To confirm in vitro mimicking of tumor metastasis, a change in the levelor localization of a marker of tumor metastasis can be compared betweenthe method of the invention and the same method in the absence of theapplication of the shear stress. The level or localization of the markerin the at least one tumor cell type upon application of the shear stressis compared to the level or localization of the marker in the at leastone tumor cell type in the absence of the application of the shearstress. Alternatively, the level of marker in the culture medium uponapplication of the shear stress is compared to the level of the markerin the culture medium upon the absence of the application of shearstress. For example, if a marker is known to be associated with tumormetastasis, and its concentration is known to increase in the serumduring metastasis in vivo, an increase in the level of the marker in theculture medium of the method of the invention with application of shearstress as compared to the level of the marker in the culture medium inthe absence of the application of the shear stress confirms that tumormetastasis is mimicked by the in vitro method of the invention.

Methods for Mimicking Tumor Metastasis in an Animal

The present invention also provides a method for mimicking tumormetastasis. The method comprises introducing cells of the at least onetumor cell type cultured according to any of the methods described aboveinto an animal. The animal can be a mammal, for example a mouse, rat,guinea pig, hamster rabbit, cat, dog, monkey, cow, pig, horse, goat, orsheep. Alternatively, the animal can be a bird or a fish.

Where the animal is a mouse, the mouse is suitably a humanized mouse,and the at least one tumor cell type comprises a human tumor cell type.The humanized mouse can be a non-obese diabetic severe combinedimmunodeficiency (NOD SCID) mouse, a NOD/Shi-scid/IL-2Rγnull (NOG)mouse, or a NOD SCID IL-2Rγ knockout (NSG) mouse.

Personalized Medicine

The present invention provides a method for selecting a chemotherapyregimen to be administered to a subject having a tumor. The methodcomprises testing a drug or a compound in vitro for an effect on a tumoror testing a drug or a compound for an in vitro effect on tumormetastasis according to any of the methods described herein. The atleast one tumor cell type comprises tumor cells derived from thesubject's tumor. The method further comprises determining whether toadminister the drug or the compound to the subject based on the resultsof the in vitro testing.

The method can further comprise selecting a dose of the drug or thecompound to be administered to the subject based on the results of thein vitro testing. The dose selected will be a dose that is predicted tobe both therapeutic and safe in the subject based on the results of thein vitro testing.

The method can further comprise selecting a rate of administration ofthe drug or the compound to be administered to the subject based on theresults of the in vitro testing. The rate selected will be a rate thatis predicted to be both therapeutic and safe in the subject based on theresults of the in vitro testing.

Drugs and Compounds

In any of the in vitro methods of testing a drug or a compound for aneffect on a tumor or for an effect on tumor metastasis described herein,the at least one tumor cell type can be exposed to the drug or thecompound directly or indirectly. The at least one tumor cell type can bedirectly exposed to the drug or compound by adding the drug or compoundto cell culture medium containing or contacting the at least one tumorcell type. For example, where the at least one tumor cell type is platedon a first surface of a porous membrane, and the porous membrane issuspended in the cell culture container such that the first surface isproximal and in spaced relation to a bottom surface of the cell culturecontainer, thereby defining within the cell culture container a lowervolume comprising the at least one tumor cell type and an upper volumecomprising a second surface of the porous membrane, the at least onetumor cell type can be directly exposed to the drug or compound byadding the drug or compound to the cell culture medium in the lowervolume. Adding the drug or compound to the upper volume can also resultin directly exposing the at least one tumor cell type to the drug orcompound, for example where the drug or compound is small enough todiffuse through the pores of the membrane or where the tumor cells havemigrated into the upper volume. Alternatively, the at least one tumorcell type can be indirectly exposed to the drug or compound. Forexample, the at least one tumor cell type would be indirectly exposed tothe drug or the compound when the drug or the compound is added to theupper volume and the drug or compound does not diffuse through the poresof the membrane, but exerts an effect on endothelial cells plated on thesecond surface of the porous membrane which in turn causes theendothelial cells to exert an effect on the at least one tumor cell type(e.g., secretion of a factor by the endothelial cells that diffusesthrough the porous membrane and has an effect on the at least one tumorcell type, or physical interaction of the endothelial cells with the atleast one tumor cell type through the pores of the porous membrane).

The concentration of the drug or compound in the culture medium can bewithin the concentration range of the drug or the compound that achievesthe effect in vivo. For example, the concentration of the drug or thecompound in the culture medium can be within the concentration range ofthe in vivo therapeutic C_(max) for the drug or the compound (e.g., thein vivo therapeutic plasma C_(max) for the drug or the compound). Theconcentration of the drug or the compound in the culture medium can beapproximately the same as the in vivo therapeutic C_(max) for the drugor the compound (e.g., the in vivo therapeutic plasma C_(max) for thedrug or the compound).

Alternatively, the concentration of the drug or the compound in theculture medium can be lower than the concentration range of the drug orthe compound that achieves the effect in vivo. This mimics the lowerdegree of drug penetration that is observed in many solid tumors invivo. For example, the concentration of the drug or the compound in theculture medium can be about 2-fold to about 20-fold lower, about 5-foldto about 15-fold lower, or about 10-fold lower than the concentrationrange of the in vivo therapeutic C_(max) for the drug or the compound(e.g., the in vivo therapeutic plasma C_(max) for the drug or thecompound).

The effect of the drug or the compound can comprise a toxic effect, aprotective effect, a pathologic effect, a disease-promoting effect, aninflammatory effect, an oxidative effect, an endoplasmic reticulumstress effect, a mitochondrial stress effect, an apoptotic effect, anecrotic effect, an autophagic effect, an immunogenic cell death effect,a ferroptotic effect, a remodeling effect, a proliferative effect, aneffect on angiogenesis, an effect on the activity of a protein, or aneffect on the expression of a gene. The term “proliferative effect”encompasses both stimulation of proliferation and inhibition ofproliferation. Similarly, the effect on angiogenesis encompasses bothstimulation of angiogenesis and inhibition of angiogenesis.

Where the effect comprises the effect on the activity of a protein, theeffect can comprise inhibition of the protein or activation of theprotein.

Where the effect comprises the effect on the expression of a gene, theeffect can comprise an increase in the expression of the gene or adecrease in the expression of the gene.

The in vitro methods of testing a drug or a compound for an effect on atumor or on tumor metastasis can be used to screen candidate moleculesfor anti-cancer activity.

The in vitro methods of testing a drug or a compound for an effect on atumor or on tumor metastasis can also be used to test drugs or compoundsknown or suspected to have anti-cancer activity.

The drug or compound can be capable of inhibiting, activating, oraltering the function of proteins or genes in the at least one celltype.

The drug can comprise an anti-cancer agent. Anti-cancer agents include,for example, alkylating agents, anti-metabolites, anti-tumorantibiotics, topoisomerase inhibitors, corticosteroids, anti-microtubuleagents, kinase inhibitors, pathway inhibitors, differentiating agents,hormone therapies, immunotherapies, L-asparaginase, chelating agents,ATP mimetics, biologic medical products, and combinations thereof.

When the anti-cancer agent comprises the alkylating agent, thealkylating agent can comprise altretamine, bendamustine, busulfan,carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide,dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan,oxalaplatin, palifosamide, streptozocin, temozolomide, thiotepa, or acombination thereof.

When the anti-cancer agent comprises the anti-metabolite, theantimetabolite can comprise azathioprine, capecitabine, cladribine,clofarabine, cytarabine, floxuridine, fludarabine, fluorouracil,gemcitabine, hydroxyurea, mercaptopurine, methotrexate, nelarabine,pemetrexed, pentostatin, pralatrexate, raltitrexed, thioguanine, or acombination thereof.

When the anti-cancer agent comprises the anti-tumor antibiotic, theanti-tumor antibiotic can comprise bleomycin, dactinomycin, mitomycin,plicamycin, rifampicin, or a combination thereof.

When the anti-cancer agent comprises the topoisomerase inhibitor, thetopoisomerase inhibitor can comprise amsacrine, topotecan, irinotecan,etoposide, teniposide, mitoxantrone, etirinotecan, camptothecin,daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin,amonafide, or a combination thereof.

When the anti-cancer agent comprises the corticosteroid, thecorticosteroid can comprise prednisone, methylprednisolone,dexamethasone, cortisol sodium succinate, or a combination thereof.

When the anti-cancer agent comprises the anti-microtubule agent, theanti-microtubule agent can comprise vinblastine, vincristine, vindesine,vinorelbine, paclitaxel, docetaxel, ixabepilone, eribulin mesylate,cabazitaxel, or a combination thereof.

When the anti-cancer agent comprises the kinase inhibitor, the kinaseinhibitor can comprise a small molecule inhibitor of a receptor ornon-receptor tyrosine kinase, a serine/threonine-specific kinaseinhibitor, or a dual-specificity kinase inhibitor.

The kinase inhibitor can comprise an epidermal growth factor (EGF)receptor inhibitor, a fibroblast growth factor (FGF) receptor inhibitor,a platelet-derived growth factor (PDGF) receptor inhibitor, a vascularendothelial growth factor (VEGF) receptor inhibitor, or a rho kinaseinhibitor.

When the kinase inhibitor comprises the small molecule inhibitor of areceptor or non-receptor tyrosine kinase, the small molecule inhibitorof a receptor or non-receptor tyrosine kinase can comprise afatinib,alectinib, alisertib, amuvatinib, apatinib, axitinib, bafetinib,barasertib, baricitinib, bosutinib, brivanib, buparlisib, cabozantinib,canertinib, cenisertib, cobimetinib, crenolanib, crizotinib, dabrafenib,dacomitinib, danusertib, desatinib, dovitinib, epitinib, erlotinib,foretinib, fostamatinib, galunisertib, gefitinib, ibrutinib, imatinib,lapatinib, lenvatinib, lestaurtinib, linifanib, linsitinib, masitinib,momelotinib, motesanib, mubritinib, neratinib, nilotinib, nintedanib,orantinib, pacritinib, pazopanib, pelitinib, pimasertib, ponatinib,poziotinib, quizartinib, refametinib, regorafenib, ruxolitinib,selumetanib, sorafenib, sulfatinib, sunitinib, tandutinib, telatinib,theliatinib, tivantinib, tofacitinib, trametinib, vandetanib, vatalinib,vemurafenib, volasertib, volitinib, or a combination thereof.

When the kinase inhibitor comprises the serine/threonine-specific kinaseinhibitor, the serine/threonine-specific kinase inhibitor can compriseMK2206.

When the anti-cancer agent comprises the pathway inhibitor, the pathwayinhibitor can comprise a B-cell lymphoma 2 (Bcl-2) family inhibitor(e.g., navitoclax, obatoclax, oblimerson, or cinacalcet), a heat shockprotein 90 (HSP-90) inhibitor (e.g., tanespimycin, retaspimycin, organetespib), a proteasome inhibitor (e.g., bortezomib, carfilzomib,oprozomib, ixazomib, marozomib, or delanzomib), a cyclin-dependentkinase inhibitor (e.g., flavopiridol, alvocidib, dinaciclib, seliciclib,or palbociclib), an inhibitor of poly ADP-ribose polymerase (PARP)(e.g., iniparib, veliparib, olaparib, rucaparib, or niraparib), aninhibitor of the mammalian target of rapamycin (mTOR) (e.g.,deforolimus, everolimus, sirolimus, or temsirolimus), an inhibitor ofhistone deacetylase (HDAC) (e.g., belinostat, entinostat, mocetinostat,panobinostat, romidepsin, or vorinostat), an inhibitor of the hedgehogpathway (e.g., varidegib or vismodegib), a rho kinase inhibitor (e.g.,Y27632), or a combination thereof.

When the anti-cancer agent comprises the differentiating agent, thedifferentiating agent can comprise a retinoid, tretinoin, bexarotene,arsenic trioxide, or a combination thereof.

When the anti-cancer agent comprises the hormone therapy, the hormonetherapy can comprise a selective androgen-receptor modulator (SARM)(e.g., enobosarm), an androgen receptor antagonist (e.g., bicalutamide,flutamide, nilutamide, or enzalutamide), a selective estrogen receptormodulator (SERM) (e.g., tamoxifen, toremifene, or raloxifene), anestrogen receptor antagonist (e.g., fulvestrant), a progestin (e.g.,megestrol acetate), an estrogen (e.g., estramustine), an aromataseinhibitor (e.g., anastrozole, exemestane, or letrozole), agonadotropin-releasing hormone (GnRH) agonist or analog (e.g.,leuprolide, goserelin, abarelix, degarelix, or triptorelin),ketoconazole, abiraterone, or a combination thereof.

When the anti-cancer agent comprises the immunotherapy, theimmunotherapy can comprise a monoclonal antibody (e.g., rituximab,alemtuzumab, bevacizumab, abagovomab, or etaracizumab), a non-specificimmunotherapy or adjuvant (e.g., interleukin-2 (IL-2), interferon-α,interferon-α2b, peginterferon alfa-2b, abatacept, or aldesleukin), animmunomodulating drug (e.g., thalidomide or lenalidomide), a cancervaccine (e.g., Sipuleucel-T or Bacillus Calmette-Guérin (BCG) vaccine),a targeted immunotherapy (e.g., brentuzimab, cetuximab, ibritumomab,ipilimumab, ofatumumab, panitumumab, pertuzumab, tositmuomab,trastuzumab, tremelimumab, siltuximab, tocilizumab, canakinumab,lirilumab, nivolumab, pidilizumab, or lambrolizumab), or a combinationthereof.

When the anti-cancer agent comprises the chelating agent, the chelatingagent can comprise penicillamine, triethylene tetramine dihydrochloride,EDTA, DMSA, deferoxamine mesylate, or batimastat.

When the anti-cancer agent comprises the biologic medical product, thebiologic medical product can comprise a synthetic polysaccharide; asynthetic, partially synthetic or humanized immunoglobulin; or arecombinant therapeutic protein.

The drug or the compound can comprise a radiocontrast agent, aradio-isotope, a prodrug, an antibody fragment, an antibody, a livecell, a therapeutic drug delivery microsphere, microbead, nanoparticle,gel or cell-impregnated gel, or a combination thereof.

In any of the in vitro methods of testing a drug or a compound for aneffect on a tumor or for an effect on tumor metastasis described herein,adding the drug or the compound to the culture medium can compriseadding an antibody-drug conjugate or a modified release dosage formcomprising the drug or the compound to the culture medium.

The modified release dosage form can comprise an oral modified releasedosage form.

The modified release dosage form can a modified release polymer (e.g.,hydroxypropylcellulose, hydroxypropylmethylcellulose,hydroxyethylcellulose, ethylcellulose, methylcellulose,carboxymethylcellulose, alginic acid, carrageenan, chitosan, heparin,starch, xanthan gum, polyvinyl alcohol, polyacrylic acid, polyethyleneoxide, poloxamers, pluronics, polymethacrylate, polysialic acid, or acombination thereof).

The method can comprise perfusing the drug or the compound into at leastone of the upper volume and the lower volume.

Analysis of the Cell Types and Cell Culture Medium

In methods of the invention involving comparing a change in the level orlocalization of a marker of the tumor microenvironment or a marker oftumor metastasis between a method of the invention and the same methodin the absence of the application of the shear stress, the marker cancomprise a marker of cell proliferation, cell invasion, angiogenesis,tumorigenesis, cell monolayer integrity, endothelial cell barrierfunction, permeability, inflammation, cell death, apoptosis, necrosis,contraction, cell motility, or a combination thereof.

The change in the level of a marker can be an increase in the level ofthe marker in the at least one tumor cell type or the endothelial cells.

The change in the level of a marker can be a decrease in the level ofthe marker in the at least one tumor cell type or the endothelial cells.

The marker can comprise VE-cadherin, E-cadherin, actin, or a combinationthereof.

When the marker comprises a marker of angiogenesis, the marker ofangiogenesis can comprise vascular endothelial growth factor (VEGF)-A,VEGF-C, VEGF-D, angiopoietin-1 (ANG1), angiopoietin-2 (ANG2), fibroblastgrowth factor-2 (FGF-2), placental growth factor (PLGF), or acombination thereof.

When the marker comprises a marker of cell proliferation, the marker ofcell proliferation can comprise epidermal growth factor (EGF), epidermalgrowth factor receptor (EGFR), MKI67, proliferating cell nuclear antigen(PCNA), or a combination thereof.

When the marker comprises a marker of cell invasion, the marker of cellinvasion can comprise vimentin (VIM), cadherin 1 (CDH-1), cadherin 2(CDH-2), or a combination thereof.

When the marker comprises a marker of inflammation, the marker ofinflammation can comprises interleukin-6 (IL-6), interleukin-8 (IL-8),NF-κB, endothelial nitric oxide synthase (eNOS), Krupple-like factor 2(KLF2), monocyte chemotactic protein-1 (MCP-1), or a combinationthereof.

The methods described herein can further comprise analyzing theendothelial cells for cell density, monolayer integrity, permeability,or a combination thereof.

The methods described herein can also further comprise analyzing themorphology of the at least one tumor cell type, the endothelial cells,the at least one stromal cell type, or the one or more additional celltypes.

The methods described herein can also further comprise analyzing theculture medium for cytokine secretion, chemokine secretion, humoralfactor secretion, microparticle secretion, growth factor secretion,shedding of a protein from the cellular surface, a metabolite of acompound, an immune cell, nitric oxide secretion, a vasodilator protein,a vasoconstrictive protein, miRNA, a secreted protein, or a secretedbiological substance. For example, the culture medium can be analyzedfor shedding of a protein from the cellular surface, and the proteincomprises a vascular cell adhesion molecule (VCAM), E-selectin, or anintracellular adhesion molecule (ICAM). Alternatively or in addition,the culture medium can be analyzed for nitric oxide secretion bymeasuring nitrate or nitrite concentration.

In any of the methods comprising adding a drug or a compound to theculture medium, the method can further comprise analyzing the at leastone tumor cell type, the endothelial cells, the at least one stromalcell type for toxicity, or the one or more additional cell types forinflammation, permeability, compatibility, cellular adhesion, cellularremodeling, cellular migration, or phenotypic modulation resulting fromthe drug or the compound.

Also, in any of the methods comprising adding a drug or a compound tothe culture medium, the method can further comprise comparing at leastone of the cell types after applying the shear stress for a period oftime wherein the medium includes the drug or the compound to the atleast one of the cell types after applying the shear stress for theperiod of time wherein the medium does not include the drug or thecompound, to determine the effect of the drug or compound on the atleast one of the cell types.

Any of the methods described herein can further include identifying adrug target. For example, a drug target can be identified by isolatingproteins or nucleic acids from the at least one tumor cell type directlyor indirectly exposed to the drug or compound and performing anappropriate screen to identify potential drug targets. Screening methodsinclude proteomic analysis or phosphorylation screening, mRNA analysis(e.g., next generation RNA sequencing or gene arrays), DNA analysis, DNAmethylation screening, and intracellular or extracellular miRNA analysis(e.g., miRNA arrays). Modulation of a signal (e.g., increased ordecreased expression of a gene) indicates identification of a candidatedrug target.

Any of the methods described herein can further include identifying asurface protein of the at least one tumor cell type, the at least onestromal cell type, the endothelial cells, or the one or more additionalcell types as a target for a drug delivery modality. The drug deliverymodality can comprise an antibody-drug conjugate, a nanoparticle (e.g.,a lipid nanoparticle), a chemical conjugate (e.g., N-Acetylgalactosamine(GalNAc)), or a combination thereof. A protein, antibody, peptide, ornucleic acid molecule (e.g., an RNAi molecule) can be conjugated to orincorporated in the nanoparticle or the chemical conjugate. Surfaceproteins that are targets for a drug delivery modality can be identifiedby isolating the cell membrane fraction from tumor cells, stromal cells,endothelial cells, or the one or more additional cell types culturedaccording to any of the methods for mimicking a tumor microenvironmentdescribed herein, screening the cell membrane faction to identifypotential targets for a drug delivery modality. Screening methodsinclude proteomic analysis or phosphorylation screening, mRNA analysis(e.g., next generation RNA sequencing or gene arrays), DNA analysis, DNAmethylation screening, and intracellular or extracellular miRNA analysis(e.g., miRNA arrays). Modulation of a signal (e.g., increased ordecreased expression of a gene) indicates identification of a candidatetarget for a drug delivery modality.

In Vitro Systems that Model the Liver

As noted above, in vitro systems that model the liver are described inU.S. Patent Application Publication No. 2013/0309677 and PCT PublicationNo. 2013/0158939. The in vitro systems that model the liver described inthese publications can be used in method for mimicking an in vivopathological or physiologic condition. Unlike static models currentlyused as the standard in vitro models by the pharmaceutical andbiopharmaceutical industries, the methods described in U.S. PatentApplication Publication No. 2013/0309677 and PCT Publication No.2013/0158939 apply shear forces to cultured cells and replicate an invivo pathological or physiological condition using in vivo pathologicalor physiologic concentrations of various factors. For example, an invitro liver model is described in which hepatocytes can be maintained atin vivo physiologic concentrations of insulin and glucose that aresignificantly decreased as compared to the concentrations used in thestandard static model. When higher concentrations of insulin and glucoseare used in such a model, the hepatocytes exhibit numerous hallmarks offatty liver disease.

U.S. Patent Application Publication No. 2013/0309677 and PCT PublicationNo. 2013/0158939 describe a method for mimicking a pathologicalcondition in vitro (e.g., a pathological condition of the liver). Themethod comprises adding a culture media to a cell culture container,adding at least one factor to the culture media, plating at least onecell type on at least one surface within the cell culture container, andapplying a shear force upon the at least one plated cell type. The shearforce results from flow of the culture media induced by a flow device.The flow mimics flow to which the at least one cell type is exposed invivo in the pathological condition.

The concentration of the factor in the culture media can be within thein vivo concentration range of the factor observed in the pathologicalcondition. Alternatively, the concentration of the factor in the culturemedia can be within the concentration range of the factor that wouldresult in vivo from administration of a drug or a compound.

To confirm that the in vivo pathological condition is mimicked, a changein a level of a marker of the pathological condition can be comparedbetween the method and the same method in the absence of application ofthe shear force. The level of the marker in the at least one plated celltype or in the culture media upon application of the shear force iscompared to the level of the marker in the at least one plated cell typeor in the culture media in the absence of application of the shearforce. For example, if a marker is known to be associated with apathological condition and its concentration is known to increase in theserum when the condition is present in vivo, an increase in the level ofthe marker in the culture media of the method with application of theshear force as compared to the level of the marker in the culture mediain the absence of application of the shear force confirms that the invivo pathological condition is mimicked by the in vitro method.

U.S. Patent Application Publication No. 2013/0309677 and PCT PublicationNo. 2013/0158939 also describe an in vitro method of testing a drug or acompound for an effect on a pathological condition. The method comprisesmimicking the pathological condition, adding a drug or a compound to theculture media, and applying the shear force upon the at least one platedcell type exposed to the drug or the compound. A change in the at leastone plated cell type, in the presence of the drug or the compound,indicates that the drug or the compound has an effect on thepathological condition.

In this in vitro method of testing a drug or compound, the pathologicalcondition can be mimicked by the in vitro method of mimicking apathological condition as described above.

The pathological condition of the in vitro method of testing a drug orcompound can also be mimicked by plating primary cells or immortalizedcells from a subject or subjects having the pathological condition, andculturing the cells in cell culture media.

U.S. Patent Application Publication No. 2013/0309677 and PCT PublicationNo. 2013/0158939 also describe a method of mimicking a physiologiccondition in vitro (e.g., a healthy liver). The method comprises addinga culture media to a cell culture container, adding at least one factorto the culture media, plating at least one cell type on at least onesurface within the cell culture container, and applying a shear forceupon the at least one plated cell type. The shear force results fromflow of the culture media induced by a flow device. The flow mimics flowto which the at least one cell type is exposed in vivo in thephysiologic condition.

The concentration of the factor in the culture media can be within thein vivo concentration range of the factor observed in the physiologiccondition. Alternatively, the concentration of the factor in the culturemedia can be within the concentration range of the factor that wouldresult in vivo from administration of a drug or a compound.

To confirm that the in vivo physiologic condition is mimicked, a changein a level of a marker of the physiologic condition can be comparedbetween the method and the same method in the absence of application ofthe shear force. The level of the marker in the at least one plated celltype or in the culture media upon application of the shear force iscompared to the level of the marker in the at least one plated cell typeor in the culture media in the absence of application of the shearforce. For example, if a marker is known to be associated with aphysiologic condition and its concentration is known to increase in theserum when the condition is present in vivo, an increase in the level ofthe marker in the culture media of the method with application of theshear force as compared to the level of the marker in the culture mediain the absence of application of the shear force confirms that the invivo physiologic condition is mimicked by the in vitro method.

U.S. Patent Application Publication No. 2013/0309677 and PCT PublicationNo. 2013/0158939 also describe an in vitro method of testing a drug or acompound for an effect on a physiologic condition. The method comprisesmimicking the physiologic condition, adding a drug or a compound to theculture media, and applying the shear force upon the at least one platedcell type exposed to the drug or the compound. A change in the at leastone plated cell type, in the presence of the drug or the compound,indicates that the drug or the compound has an effect on the physiologiccondition.

In this in vitro method of testing a drug or compound, the physiologiccondition can be mimicked by the in vitro method of mimicking aphysiologic condition as described above.

The physiologic condition of this in vitro method of testing a drug orcompound can also be mimicked by plating primary cells or immortalizedcells, and culturing the cells in cell culture media.

U.S. Patent Application Publication No. 2013/0309677 and PCT PublicationNo. 2013/0158939 also describe an in vitro method of testing a drug or acompound for an effect (e.g., an effect on a pathological condition ofthe liver or on a healthy liver). The method comprises adding a culturemedia to a cell culture container, plating at least one cell type on atleast one surface within the cell culture container, adding a drug or acompound to the culture media, and applying a shear force upon the atleast one plated cell type exposed to the drug or the compound. Theconcentration of the drug or the compound in the culture media is withinthe concentration range of the drug or the compound that achieves theeffect in vivo. The shear force results from flow of the culture mediainduced by a flow device. The flow mimics flow to which the at least onecell type is exposed in vivo. A change in the at least one plated celltype, in the presence of the drug or the compound, indicates that thedrug or the compound has the effect.

The effect can be an effect on a pathological condition (e.g., apathological condition of the liver). Alternatively, the effect can bean effect on a physiologic condition (e.g., a healthy liver.

In any of the above methods described in U.S. Patent ApplicationPublication No. 2013/0309677 and PCT Publication No. 2013/0158939, themethod can further comprise analyzing the cell culture media forcytokine secretion, chemokine secretion, humoral factor secretion,microparticle secretion, growth factor secretion, shedding of a proteinfrom the cellular surface, a metabolite of a compound, an immune cell,nitric oxide secretion, a vasodilator protein, a vasoconstrictiveprotein, miRNA, a secreted protein, or a secreted biological substance.The cell culture media can be analyzed for nitric oxide secretion bymeasuring nitrate or nitrite concentration.

In any of the above methods described in U.S. Patent ApplicationPublication No. 2013/0309677 and PCT Publication No. 2013/0158939, themethod can further comprise the step of culturing the cell type or celltypes.

In any of the above methods described in U.S. Patent ApplicationPublication No. 2013/0309677 and PCT Publication No. 2013/0158939wherein a drug or compound has been added to the culture media, themethod can further comprise the step of comparing at least one of thecell types after applying the shear force for a period of time whereinthe media includes the drug or the compound to the at least one of thecell types after applying the shear force for the period of time whereinthe media does not include the drug or the compound, to determine theeffect of the drug or compound on the at least one of the cell types.

U.S. Patent Application Publication No. 2013/0309677 and PCT PublicationNo. 2013/0158939 describe methods wherein drugs or compounds are testedfor an effect on a healthy liver. In such methods, the factors compriseinsulin and glucose, hepatocytes are plated on the surface within thecell culture container, and the shear force is applied indirectly to theplated hepatocytes.

For example, the hepatocytes can be plated on a first surface of aporous membrane. The porous membrane is then suspended in the cellculture container such that the first surface is proximal and in spacedrelation to a bottom surface of the cell culture container, therebydefining within the cell culture container a lower volume and an uppervolume. The lower volume comprises the hepatocytes and the upper volumecomprises a second surface of the porous membrane. The shear force isapplied to the second surface of the porous membrane in the upper volumeof the container.

In the methods described in U.S. Patent Application Publication No.2013/0309677 and PCT Publication No. 2013/0158939, use of a porousmembrane suspended in the cell culture container is preferred in platingthe cells. When shear force is applied to plated cells or to the surfaceof the porous membrane (e.g., when the shear is applied on a surface ofthe membrane absent plated cells), the shear force can enable the cellculture media to perfuse from the upper volume to the lower volume. Suchperfusion favorably impacts transport of factors from the upper volumeto the lower volume, or vice versa.

U.S. Patent Application Publication No. 2013/0309677 and PCT PublicationNo. 2013/0158939 describe methods of mimicking a pathological orphysiologic condition of the liver in vitro. The method comprises addinga culture media to a cell culture container, adding at least one factorto the culture media, plating at least one hepatic cell type on at leastone surface within the cell culture container, and applying a shearforce upon the at least one plated hepatic cell type. The shear forceresults from flow of the culture media induced by a flow device. Theflow mimics flow to which the at least one hepatic cell type is exposedin vivo in the pathological or physiologic condition.

In this method, the concentration of the factor in the culture media formimicking the pathological condition can be within the in vivoconcentration range of the factor observed in the pathologicalcondition. Alternatively, in this method, the concentration of thefactor in the culture media for mimicking the pathological condition canbe within the concentration range of the factor that would result invivo from administration of a drug or a compound. As a furtheralternative, in this method, the concentration of the factor in theculture media for mimicking the pathological condition can be capable ofmaintaining the mimicked pathological condition in vitro for a period oftime under the shear force, the same concentration of factor beingincapable of maintaining the mimicked pathological condition in vitrofor the period of time in the absence of the shear force.

In this method, the concentration of the factor in the culture media formimicking the physiologic condition can be within the in vivoconcentration range of the factor observed in the physiologic condition.Alternatively, in this method, the concentration of the factor in theculture media for mimicking the physiologic condition can be within theconcentration range of the factor that would result in vivo fromadministration of a drug or a compound. As a further alternative, inthis method, the concentration of the factor in the culture media formimicking the physiologic condition can be capable of maintaining themimicked physiologic condition in vitro for a period of time under theshear force, the same concentration of factor being incapable ofmaintaining the mimicked physiologic condition in vitro for the periodof time in the absence of the shear force.

In this method, a change in a level of a marker of the pathological orphysiologic condition in the at least one plated hepatic cell type or inthe culture media upon application of the shear force, as compared tothe level of the marker in the at least one plated hepatic cell type orin the culture media in the absence of application of the shear forceconfirms mimicking of the pathological or physiologic condition.

Alternatively, in this method, the at least one plated hepatic cell typecan comprise hepatocytes, and responsiveness to glucagon, insulin, or aglucose substrate in the hepatocytes confirms mimicking of thephysiologic condition. The glucose substrate can be, for example,glycerol, lactate, pyruvate, or combinations thereof (e.g., acombination of lactate and pyruvate).

U.S. Patent Application Publication No. 2013/0309677 and PCT PublicationNo. 2013/0158939 also describe an in vitro method of testing a drug or acompound for an effect on a pathological or physiological condition(e.g., a pathological or physiological condition of the liver). Themethod comprises mimicking the pathological or physiological condition,adding a drug or a compound to the culture media, and applying the shearforce upon at least one plated hepatic cell type exposed to the drug orthe compound. A change in the at least one plated hepatic cell type, inthe presence of the drug or the compound, indicates that the drug or thecompound has an effect on the pathological or physiological condition.

In this in vitro method of testing a drug or compound, the pathologicalcondition can be mimicked by the in vitro method of mimicking apathological or physiological condition as described directly above.

The pathological or physiological condition of the in vitro method oftesting a drug or compound can also be mimicked by plating primary cellsor immortalized cells from a subject or subjects having the pathologicalcondition, and culturing the cells in cell culture media.

U.S. Patent Application Publication No. 2013/0309677 and PCT PublicationNo. 2013/0158939 also describe a method of mimicking a pathological orphysiologic condition of the liver in vitro. The method comprises addinga culture media to a cell culture container, depositing at least oneextracellular matrix component on a surface within the cell culturecontainer, plating hepatocytes on the at least one extracellular matrixcomponent, and indirectly applying a shear force upon the at least oneextracellular matrix component and the hepatocytes. The shear forceresults from flow of the culture media induced by a flow device. Theflow mimics flow to which the hepatocytes are exposed in vivo in thepathological or physiologic condition.

In methods in which hepatic cells are plated on a porous membrane, atleast one extracellular matrix component can be plated on a firstsurface of the porous membrane and the hepatic cells can subsequently beplated on the at least one extracellular matrix component. Optionally,nonparenchymal hepatic cells (e.g., sinusoidal endothelial cells) can beplated on the second surface of the porous membrane, and the shearstress applied to the nonparenchymal hepatic cells.

In the methods involving the deposition of an extracellular matrixcomponent, for example, the at least one extracellular matrix componentcan be deposited on a first surface of a porous membrane. The hepaticcell type (e.g., hepatocytes) is subsequently plated on the at least oneextracellular matrix component. The porous membrane is suspended in thecell culture container such that the first surface is proximal and inspaced relation to a bottom surface of the cell culture container,thereby defining within the cell culture container a lower volume and anupper volume. The lower volume comprises at least one extracellularmatrix component and the hepatic cell type (e.g., hepatocytes), and theupper volume comprises a second surface of the porous membrane. Theshear force is applied to the second surface of the porous membrane inthe upper volume of the container. Optionally, nonparenchymal hepaticcells (e.g., sinusoidal endothelial cells) can be plated on the secondsurface of the porous membrane, and the shear stress applied to thenonparenchymal hepatic cells.

U.S. Patent Application Publication No. 2013/0309677 and PCT PublicationNo. 2013/0158939 also describe another method of mimicking apathological or physiologic condition of the liver in vitro. The methodcomprises adding a culture media to a cell culture container, andplating hepatocytes on a first surface of a porous membrane. The porousmembrane is suspended in the cell culture container such that the firstsurface is proximal and in spaced relation to a bottom surface of thecontainer, thereby defining within the container a lower volumecomprising the hepatocytes and an upper volume comprising a secondsurface of the porous membrane. A shear force is applied upon the secondsurface of the porous membrane in the upper volume of the container, theshear force resulting from flow of the culture media induced by a flowdevice. The flow mimics flow to which the hepatocytes are exposed invivo in the pathological or physiologic condition. The flow devicecomprises a body adapted for being positioned in the culture media inthe upper volume of the container and a motor adapted to rotate thebody. Preferably, the body has a conical surface. It is also preferredthat the flow device is adapted for positioning the conical surface ofthe body in the container and in contact with the cell culture media.

This method can further comprise plating nonparenchymal hepatic cells onthe second surface of the porous membrane, wherein the shear stress isapplied to the nonparenchymal hepatic cells. The nonparenchymal hepaticcells can comprise sinusoidal endothelial cells, hepatic stellate cells,Kupffer cells, or combinations thereof.

In the in vitro methods for mimicking a pathological or physiologiccondition of the liver, a change in a level of a marker of thepathological or physiologic condition can be compared in the method tothe same method in the absence of application of the shear force. Achange in the level of the marker in any of the hepatic cells or in theculture media upon application of the shear force as compared to thelevel of the marker in the hepatic cells or in the culture media in theabsence of application of the shear force confirms mimicking of thepathological or physiologic condition. For example, a change in thelevel of the marker in the hepatocytes or nonparenchymal hepatic cellsor in the culture media upon application of the shear force as comparedto the level of the marker in the hepatocytes or nonparenchymal hepaticcells or in the culture media in the absence of application of the shearforce confirms mimicking of the pathological or physiologic condition.

Alternatively, when the at least one plated hepatic cell type compriseshepatocytes, responsiveness to glucagon, insulin, or a glucose substratein the hepatocytes confirms mimicking of the physiologic condition. Theglucose substrate can be, for example, glycerol, lactate, pyruvate, orcombinations thereof (e.g., a combination of lactate and pyruvate).

Pathological Conditions

The pathological conditions of the liver that can be mimicked using themethods described in U.S. Patent Application Publication No.2013/0309677 and PCT Publication No. 2013/0158939 include, but are notlimited to fatty liver disease, hepatitis C, hepatitis B, liverfibrosis, bacterial infection, viral infection, cirrhosis, andalcohol-induced liver disease.

When the pathological condition is fatty liver disease, the cell typescan comprise hepatocytes, nonparenchymal hepatic cells, or combinationsthereof. The nonparenchymal hepatic cells can include sinusoidalendothelial cells, hepatic stellate cells, Kupffer cells, orcombinations thereof.

When the pathological condition is fatty liver disease, the flow orhemodynamic pattern can be from a normal subject, a subject having fattyliver disease, or an animal genetically modified to model fatty liverdisease.

Where the pathological condition is fatty liver disease and a porousmembrane is used, hepatocytes can be plated on a first surface of theporous membrane. The porous membrane is suspended in the cell culturecontainer such that the first surface is proximal and in spaced relationto a bottom surface of the cell culture container, thereby definingwithin the cell culture container a lower volume comprising thehepatocytes and an upper volume comprising a second surface of theporous membrane. The shear force is applied to the second surface of theporous membrane in the upper volume. Optionally, nonparenchymal hepaticcells can be plated on the second surface of the porous membrane, andthe shear force is applied to the nonparenchymal hepatic cells in theupper volume. Optionally, an extracellular matrix component can bedeposited on the first surface of the porous membrane, and subsequentlyhepatoctyes can be plated on the extracellular matrix component.

Where the pathological condition is fatty liver disease and a porousmembrane is used, nonparenchymal hepatic cells can be plated on a secondsurface of a porous membrane. The porous membrane is suspended in thecell culture container such that a first surface of the porous membraneis proximal and in spaced relation to a bottom surface of the cellculture container, thereby defining within the cell culture container alower volume comprising the first surface of the porous membrane and anupper volume comprising the nonparenchymal hepatic cells. The shearforce is applied to the nonparenchymal hepatic cells in the uppervolume. Optionally, an extracellular matrix component can be depositedon the first surface of the porous membrane, and subsequentlyhepatoctyes can be plated on the extracellular matrix component.

When the vascular pathological condition is fatty liver disease, thefactor can comprise insulin, glucose, or a combination thereof. Forexample, the factor(s) can comprise insulin; glucose; or insulin andglucose.

When the pathological condition is diabetes, the cell type can comprisepancreatic β-cells, pancreatic α-cells, or a combination thereof and thefactor can comprise insulin, glucose, or insulin and glucose.

Physiologic Conditions

The physiologic conditions that can be mimicked using the methodsdescribed in U.S. Patent Application Publication No. 2013/0309677 andPCT Publication No. 2013/0158939 include the physiologic conditionscorresponding to any pathological condition of interest. For example, aphysiologic condition corresponding to fatty liver disease can be ahealthy liver state, and a physiologic condition corresponding toatherosclerosis can be an atheroprotective state.

Flow Devices

The flow devices that can be used in the methods described in U.S.Patent Application Publication No. 2013/0309677 and PCT Publication No.2013/0158939 are the same as described hereinabove in the sectionentitled “Flow Devices.”

Hemodynamic Patterns

The hemodynamic patterns that can be used in the methods described inU.S. Patent Application Publication No. 2013/0309677 and PCT PublicationNo. 2013/0158939 can be derived from a subject or subjects having thepathological condition or a disease-promoting condition. Thedisease-promoting condition can comprise atrophy, calculi, choristoma,pathologic constriction, pathologic dilation, diverticulum, hypertrophy,polyps, prolapse, rupture, an arteriovenous fistula, or an appendage(e.g., a left atrial appendage).

The hemodynamic pattern can be derived from at least a portion of anartery, an arteriole, a vein, a venule, or an organ.

When a hemodynamic pattern is derived from at least a portion of anartery or an arteriole, the artery or arteriole can comprise a carotidartery, thoracic artery, abdominal artery, pulmonary artery, femoralartery, renal efferent artery, renal afferent artery, coronary artery,brachial artery, internal mammary artery, cerebral artery, aorta,pre-capillary arteriole, hepatic artery, anterior cerebral artery,middle cerebral artery, posterior cerebral artery, basilar artery,external carotid artery, internal carotid artery, vertebral artery,subclavian artery, aortic arch, axillary artery, internal thoracicartery, branchial artery, deep branchial artery, radial recurrentartery, superior epigastric artery, descending aorta, inferiorepigastric artery, interosseous artery, radial artery, ulnar artery,palmar carpal arch, dorsal carpal arch, superficial or deep palmar arch,digital artery, descending branch of the femoral circumflex artery,descending genicular artery, superior genicular artery, inferiorgenicular artery, anterior tibial artery, posterior tibial artery,peroneal artery, deep plantar arch, arcuate artery, common carotidartery, intercostal arteries, left or right gastric artery, celiactrunk, splenic artery, common hepatic artery, superior mesentericartery, renal artery, inferior mesenteric artery, testicularis artery,common iliac artery, internal iliac artery, external iliac artery,femoral circumflex artery, perforating branch, deep femoral artery,popliteal artery, dorsal metatarsal artery, or dorsal digital artery.

When a hemodynamic pattern is derived from at least a portion of an veinor venule, the vein or venule can comprise a post-capillary venule,saphenous vein, hepatic portal vein, superior vena cava, inferior venacava, coronary vein, Thesbian vein, superficial vein, perforator vein,systemic vein, pulmonary vein, jugular vein, sigmoid sinus, externaljugular vein, internal jugular vein, inferior thyroid vein, subclavianvein, internal thoracic vein, axillary vein, cephalic vein, branchialvein, intercostal vein, basilic vein, median cubital vein,thoracoepigastric vein, ulnar vein, median antebranchial vein, inferiorepigastric vein, deep palmar arch, superficial palmar arch, palmardigital vein, cardiac vein, inferior vena cava, hepatic vein, renalvein, abdominal vena cava, testicularis vein, common iliac vein,perforating branch, external iliac vein, internal iliac vein, externalpudendal vein, deep femoral vein, great saphenous vein, femoral vein,accessory saphenous vein, superior genicular vein, popliteal vein,inferior genicular vein, great saphenous vein, small saphenous vein,anterior or posterior tibial vein, deep plantar vein, dorsal venousarch, or dorsal digital vein.

When a hemodynamic pattern is derived from at least a portion of anorgan, the organ can comprise a liver, a kidney, a lung, a brain, apancreas, a spleen, a large intestine, a small intestine, a heart, askeletal muscle, an eye, a tongue, a reproductive organ, or an umbilicalcord. The hemodynamic pattern is preferably derived from a liver.

The hemodynamic pattern can be derived from analysis of ultrasound data.

The hemodynamic pattern can be derived from analysis of magneticresonance imaging (MRI) data.

The flow or the hemodynamic pattern can be time-variant.

The flow or the hemodynamic pattern can result from a physical changeresulting from a pathological condition.

The flow or hemodynamic pattern can be derived from a subject whereinblood flow or a hemodynamic pattern has been altered as a direct orindirect effect of administration of a drug to a subject as compared tothe flow or the hemodynamic pattern for the subject absentadministration of the drug.

The flow or the hemodynamic pattern can be derived from an animal, suchas a genetically modified animal or a human. Preferably, the pattern isderived from a human.

Cell Types

The cell types that can be used in the methods described in U.S. PatentApplication Publication No. 2013/0309677 and PCT Publication No.2013/0158939 include primary cells and immortalized cells. The primarycells or immortalized cells can comprise cells isolated from at leastone subject having the pathological or physiologic condition, cellsisolated from at least one subject having a risk factor for thepathological condition, cells isolated from at least one subject with asingle nucleotide polymorphism linked to a pathological condition, cellsisolated from at least one subject with an identified genotype linked todrug toxicity, or cells isolated from at least one subject with a singlenucleotide polymorphism linked to drug toxicity.

The primary cells or the immortalized cells used in in vitro methodsinvolving a physiologic condition comprise cells isolated from at leastone subject having the physiologic condition, cells isolated from atleast one subject having a risk factor for a pathological condition,cells isolated from at least one subject with a single nucleotidepolymorphism linked to a pathological condition, cells isolated from atleast one subject with an identified genotype linked to drug toxicity,or cells isolated from at least one subject with a single nucleotidepolymorphism linked to drug toxicity.

The primary cells or immortalized cells used in in vitro methodsinvolving a pathological condition can comprise cells isolated from atleast one subject having the pathological condition, cells isolated fromat least one subject having a risk factor for the pathologicalcondition, cells isolated from at least one subject with a singlenucleotide polymorphism linked to the pathological condition, cellsisolated from at least one subject with an identified genotype linked todrug toxicity, or cells isolated from at least one subject with a singlenucleotide polymorphism linked to drug toxicity.

The primary cells or immortalized cells used in in vitro methodsinvolving a pathological condition can comprise cells isolated from atleast one subject not having the pathological condition, cells isolatedfrom at least one subject not having a risk factor for the pathologicalcondition, cells isolated from at least one subject without a singlenucleotide polymorphism linked to the pathological condition, cellsisolated from at least one subject without an identified genotype linkedto drug toxicity, or cells isolated from at least one subject without asingle nucleotide polymorphism linked to drug toxicity.

The primary cells or immortalized cells used in in vitro methodsinvolving a pathological condition can comprise cells isolated from atleast one subject having a different pathological condition, cellsisolated from at least one subject having a risk factor for a differentpathological condition, or cells isolated from at least one subject witha single nucleotide polymorphism linked to a different pathologicalcondition.

When the cells are isolated from at least one subject having a riskfactor for the pathological condition, the risk factor can include, butis not limited to, smoking, age, gender, race, epigenetic imprinting, anidentified genotype linked to the pathological condition, an identifiedsingle nucleotide polymorphism linked to the pathological condition,diabetes, hypertension, atherosclerosis, atherosclerotic plaque rupture,atherosclerotic plaque erosion, thoracic aortic aneurysm, cerebralaneurysm, abdominal aortic aneurysm, cerebral aneurysm, heart failure,stroke, Marfan syndrome, carotid intima-medial thickening, atrialfibrillation, kidney disease, pulmonary fibrosis, chronic obstructivepulmonary disease, pulmonary artery disease, pulmonary hypertension,hyperlipidemia, familial hypercholesterolemia, peripheral arterydisease, arterial thrombosis, venous thrombosis (e.g., deep veinthrombosis), vascular restenosis, vascular calcification, myocardialinfarction, obesity, hypertriglyceridemia, hypoalphalipoproteinemia,fatty liver disease, hepatitis C, hepatitis B, liver fibrosis, bacterialinfection, viral infection, cirrhosis, liver fibrosis, oralcohol-induced liver disease.

The primary cells can include a cell lineage derived from stem cells(e.g., adult stem cells, embryonic stem cells, inducible pluripotentstem cells, or bone marrow-derived stem cells) or stem-like cells. Thecell lineage derived from stem cells or stem-like cells can compriseendothelial cells, smooth muscle cells, cardiac myocytes, hepatocytes,neuronal cells, endocrine cells, pancreatic β-cells, pancreacticα-cells, or skeletal muscle cells.

The primary cells can comprise inducible pluripotent stem cell(iPSC)-derived cells from a subject having a pathological condition. Forexample, the iPSC-derived cells from a subject having a pathologicalcondition can comprise iPSC-derived hepatocytes from a subject havingfamilial hypercholesterolemia, glycogen storage disease type I, Wilson'sdisease, A1 anti-trypsin deficiency, Crigler-Najjar syndrome,progressive familial hereditary cholestasis, or hereditary tyrosinemiaType 1. Alternatively, the iPSC-derived cells from a subject having apathological condition can comprise iPSC-derived vascular cells (e.g.,iPSC-derived smooth muscle cells, iPSC-derived endothelial cells, oriPSC-derived endocardial cells) from a subject having Hutchinson-Gilfordprogeria, Williams-Beuren syndrome, Fabry's disease, Susac's syndrome,systemic capillary leak syndrome, Gleich syndrome, intravascularpapillary endothelial hyperplasia, sickle cell disease, or hepaticveno-occlusive disease.

Cell types for use in methods include vascular cells and hepatic cell.

Specific cell types for use in the methods include endothelial cells,hepatocytes, nonparenchymal hepatic cells, endothelial progenitor cells,stem cells, and circulating stem cells. The nonparenchymal hepatic cellsinclude hepatic stellate cells, sinusoidal endothelial cells, andKupffer cells. Preferably, the specific cell types can includeendothelial cells, hepatocytes, sinusoidal endothelial cells, or acombination thereof.

The cell types for use in the methods can be animal cell types, such ascells from a genetically modified animal. The animal cell types arepreferably human cell types. The human cell types can be selected on thebasis of age, gender, race, epigenetics, disease, nationality, thepresence or absence of one or more single nucleotide polymorphisms, arisk factor as described herein, or some other characteristic that isrelevant to the pathological or physiologic condition.

The shear force applied in the methods can be applied indirectly to theat least one plated cell type.

The shear force applied in the methods can be applied directly to the atleast one plated cell type.

The cell types, additional components such as extracellular matrixcomponent, and the porous membrane are within the culture media (i.e.,covered with culture media) in the methods.

The methods can further comprise analyzing at least one of the celltypes for toxicity, inflammation, permeability, compatibility, cellularadhesion, cellular remodeling, cellular migration, or phenotypicmodulation resulting from the drug or the compound.

Cell Culture Media

Standard cell culture media can be used in the methods described in U.S.Patent Application Publication No. 2013/0309677 and PCT Publication No.2013/0158939.

In Vivo Factor Concentrations

The physiologic in vivo concentrations of the factors for use in themethods described in U.S. Patent Application Publication No.2013/0309677 and PCT Publication No. 2013/0158939 are well known in theart, as are the methods of determining these in vivo concentrations.Methods for determining in vivo concentrations of factors are availablein the United States Pharmacopeia and in other literature.

A reported in vivo concentration range for a factor can vary dependingupon the method used for determining the range, the source from whichthe factor is obtained (e.g., whole blood or serum), the medicalcondition of the patient (i.e., whether the patient has a pathologicalcondition or physiologic condition), and time of day relative to normalsleep and eating schedule. However, it would be known to one of ordinaryskill in the art that a concentration outside an in vivo physiologicalconcentration range reported in the literature would be an in vivopathological concentration using the method reported for determining theconcentration. Likewise, a concentration below the lower endpoint orabove the upper endpoint of an in vivo pathological concentration rangereported in the literature would be an in vivo physiologic concentrationusing the method reported for determining the concentration; whether thein vivo physiologic concentration is below the lower endpoint or abovethe upper endpoint will depend upon the factor.

Extracellular Matrix Components

Extracellular matrix components for use in the methods described in U.S.Patent Application Publication No. 2013/0309677 and PCT Publication No.2013/0158939 can comprise heparan sulfate, chondroitin sulfate, keratansulfate, hyaluronic acid, a collagen, an elastin, a fibronectin, alaminin, a vitronectin, or combinations thereof. Collagen is a preferredextracellular matrix component, and is preferably the type of collagenthat is present in the in vivo environment of the cell type or celltype(s) that are plated for a particular pathological or physiologiccondition.

The extracellular matrix component can be secreted by fibroblasts,chondrocytes, or osteoblasts plated on the surface within the cellculture container.

Drugs or Compounds

The drug or compound for use in the methods described in U.S. PatentApplication Publication No. 2013/0309677 and PCT Publication No.2013/0158939 involving testing of a drug or compound can comprise anydrug or compound.

The concentration of the drug or the compound in the culture media issuitably within the concentration range of the drug or the compound thatachieves the effect in vivo. For example, the concentration of the drugor the compound in the culture media is suitably within theconcentration range of the in vivo therapeutic C_(max) for the drug orthe compound.

Sera

In any of the methods described in U.S. Patent Application PublicationNo. 2013/0309677 and PCT Publication No. 2013/0158939 that involveadding a factor to the culture media or adding a drug or compound to theculture media, the step of adding the factor to the culture media or thestep of adding the drug or a compound to the culture media can compriseadding sera from a subject to the culture media, wherein the seracomprises the factor, the drug, or the compound.

The subject can be an animal, e.g., as a genetically modified animal ora human. Preferably, the sera is derived from a human subject.

The sera can be from a subject having a physiologic condition or asubject having a pathological condition. For example, where the sera isfrom a subject that has a pathological condition, the pathologicalcondition can comprise advanced inflammation, atherosclerosis, diabeticnephropathy, diabetic neuropathy, diabetic retinopathy, hypertension,hypertensive encephalopathy, hypertensive retinopathy, fatty liverdisease, hypertension, heart failure, stroke, Marfan syndrome, carotidintima-medial thickening, atrial fibrillation, kidney disease, pulmonaryfibrosis, chronic obstructive pulmonary disease, hyperlipidemia,hypercholesterolemia, diabetes, atherosclerotic plaque rupture,atherosclerotic plaque erosion, thoracic aortic aneurysm, cerebralaneurysm, abdominal aortic aneurysm, cerebral aneurysm, pulmonary arterydisease, pulmonary hypertension, peripheral artery disease, arterialthrombosis, venous thrombosis (e.g., deep vein thrombosis), vascularrestenosis, vascular calcification, myocardial infarction, obesity,hypertriglyceridemia, hypoalphalipoproteinemia, hepatitis C, hepatitisB, liver fibrosis, bacterial infection, viral infection, cirrhosis,liver fibrosis, or alcohol-induced liver disease.

Effect on the Physiologic or Pathological Condition

In methods of testing a drug or a compound for an effect described inU.S. Patent Application Publication No. 2013/0309677 and PCT PublicationNo. 2013/0158939, the effect can comprise an effect on a physiologiccondition or an effect on a pathological condition. For example, theeffect on the physiologic condition or the pathological condition can bea toxic effect, a protective effect, a pathologic effect, adisease-promoting effect, an inflammatory effect, an oxidative effect,an endoplasmic reticulum stress effect, a mitochondrial stress effect,an apoptotic effect, a necrotic effect, a remodeling effect, aproliferative effect, an effect on the activity of a protein, such asinhibition of a protein or activation of a protein, or an effect on theexpression of a gene, such as an increase in the expression of the geneor a decrease in the expression of the gene.

Multiple Cell Type Configurations for the Flow Device

The methods described in U.S. Patent Application Publication No.2013/0309677 and PCT Publication No. 2013/0158939 can further compriseperfusing culture media, factors, drugs or compounds into and out of thecell container.

When the surface within the cell culture container comprises a porousmembrane suspended in the cell culture container, the method can furtherinclude the step of plating at least one cell type on a surface withinthe cell culture container comprising plating a first cell type on afirst surface of a porous membrane, and optionally plating a second celltype on a second surface of the porous membrane, wherein the porousmembrane is suspended in the cell culture container such that the firstsurface is proximal and in spaced relation to a bottom surface of thecell culture container, thereby defining within the cell culturecontainer a lower volume comprising the first cell type and an uppervolume comprising the optional second cell type. The porous membrane canbe adapted to permit fluid communication of the cell culture media andphysical interaction and communication between cells of the first celltype and cells of the optional second cell type. The shear force isapplied to the second cell type or the second surface of the porousmembrane in the upper volume. The method can further comprise perfusingculture media into and out of the upper volume and perfusing culturemedia into and out of the lower volume. The method can further compriseperfusing a drug or the compound into at least one of the upper volumeand the lower volume.

When the surface within the cell culture container comprises a porousmembrane suspended in the cell culture container, the method can furtherinclude the step of plating at least one cell type on a surface withinthe cell culture container comprising optionally plating a first celltype on a first surface of a porous membrane, and plating a second celltype on a second surface of the porous membrane, wherein the porousmembrane is suspended in the cell culture container such that the firstsurface is proximal and in spaced relation to a bottom surface of thecell culture container, thereby defining within the cell culturecontainer a lower volume comprising the optional first cell type and anupper volume comprising the second cell type. The porous membrane can beadapted to permit fluid communication of the cell culture media andphysical interaction and communication between cells of the optionalfirst cell type and cells of the second cell type. The shear force isapplied to the second cell type in the upper volume. The method canfurther comprise perfusing culture media into and out of the uppervolume and perfusing culture media into and out of the lower volume. Themethod can further comprise perfusing a drug or the compound into atleast one of the upper volume and the lower volume.

The inlets and outlets in the cell culture container can be within theportions of the cell culture container defining the upper and lowervolumes.

The methods described in this section can further comprise analyzing atleast one of the first cell type or the second cell type for toxicity,inflammation, permeability, compatibility, cellular adhesion, cellularremodeling, cellular migration, or phenotypic modulation resulting fromthe drug or the compound.

These methods can further comprise plating a third cell type on asurface of the container or the first surface or second surface of theporous membrane, suspending a third cell type in the culture mediawithin the upper volume, or suspending a third cell type in the culturemedia within the lower volume.

These methods can further comprise plating a fourth cell type on asurface of the container or the first or second surface of the porousmembrane, suspending a fourth cell type in the culture media within theupper volume, or suspending a fourth cell type in the culture mediawithin the lower volume.

These methods can further comprise plating a fifth cell type on asurface of the container or the first or second surface of the porousmembrane, suspending a fifth cell type in the culture media within theupper volume, or suspending a fifth cell type in the culture mediawithin the lower volume.

The first, second, third, fourth and fifth cell types can be variousprimary or immortalized cell types as described in the section aboveregarding cell types.

In each of these combinations, the cells of the third cell type, thecells of the fourth cell type or the cells of the fifth cell type can beadhered to the bottom surface of the container.

DEFINITIONS

With respect to the methods described in U.S. Patent ApplicationPublication No. 2013/0309677 and PCT Publication No. 2013/0158939, theterm “factor” means a biological substance that contributes to theproduction of a pathological or physiologic condition. Preferably, thefactor provides a change in a level of a marker of the pathological orphysiologic condition in the at least one plated cell type or in theculture media upon application of the shear force, as compared to thelevel of the marker in the at least one plated cell type or in theculture media in the absence of application of the shear force.

The term “pathological condition” means an abnormal anatomical orphysiological condition, which includes the objective or subjectivemanifestation of a disease.

The term “physiologic condition” means a normal medical state that isnot pathologic, and can be a medical state characteristic of orconforming to the normal functioning or state of the body or a tissue ororgan.

Physiologic Liver Model

The methods described in U.S. Patent Application Publication No.2013/0309677 and PCT Publication No. 2013/0158939 can be used to createa physiologic in vitro model of the liver. In such methods, hepatocytesare plated on a surface within a cell culture container, and shearforces are applied indirectly to the plated hepatocytes. For example,the hepatocytes are suitably plated on a first surface of a porousmembrane, where the porous membrane is suspended in a cell culturecontainer such that the first surface is proximal and in spaced relationto a bottom surface of the cell culture container, thereby definingwithin the cell culture container a lower volume comprising thehepatocytes and an upper volume comprising a second surface of theporous membrane. The shear force is applied to the second surface of theporous membrane in the upper volume of the container. Thus, theconfiguration of cells in the device is based on in vivomicroarchitecture of hepatic lobules.

In hepatic lobules in vivo, cords of hepatocytes are separated fromsinusoidal blood flow by a filtering layer of sinusoidal endothelialcells and a layer of extracellular matrix. The layer of extracellularmatrix provides for anchorage of the hepatocytes, is involved insignaling, and provides a reservoir of cytokines and growth factors. Thehepatocytes have a polarized morphology and biliary canaliculi arepresent in the hepatocyte layer. Sinusoidal blood flow and interstitialblood flow provide for oxygen and nutrient transport.

FIG. 11 depicts an exemplary configuration used in the in vitro livermodel and is described above. The porous membrane acts analogously tothe filtering layer of sinusoidal endothelial cells which is present inthe liver. The hepatocytes are shielded from direct effects of flow, asthey would be in vivo. Inlets and outlets in the upper and lower volumeswithin the cell culture container allow for the continuous perfusion ofculture media and for perfusion of drugs or compounds into and out ofthe cell culture media. Application of the shear force createscontrolled hemodynamics that regulate interstitial flow and solutetransfer through the porous membrane. In the in vitro models describedin U.S. Patent Application Publication No. 2013/0309677 and PCTPublication No. 2013/0158939, the hepatocytes maintain their polarizedmorphology and bile canaliculi.

At least one layer of one or more extracellular matrix components (e.g.,a collagen gel) can suitably be deposited on a first surface of theporous membrane. The hepatocytes are then plated on the extracellularmatrix component(s). One or more additional layers of the extracellularmatrix component(s) can then be deposited on top of the hepatocytes,such that the hepatocytes are substantially surrounded by theextracellular matrix component(s). The extracellular matrix componentsuitably comprises heparan sulfate, chondroitin sulfate, keratansulfate, hyaluronic acid, a collagen, an elastin, a fibronectin, alaminin, a vitronectin, or combinations thereof. For example, theextracellular matrix component can comprise collagen.

One or more additional cell types can be plated on a surface within thecell culture container or suspended in the culture media. For example,nonparenchymal hepatic cells are suitably plated on the second surfaceof the porous membrane, and the shear force is applied to the platednon-parenchymal cells. The nonparenchymal cells may include hepaticstellate cells, sinusoidal endothelial cells, Kupffer cells, orcombinations thereof. The hepatocytes and nonparenchymal hepatic cellsare suitably primary cells isolated from the liver of an animal, forexample from the liver of a human. Alternatively, the hepatocytes and/orthe nonparenchymal hepatic cells are immortalized cells.

Media is suitably continuously perfused on both sides of the porousmembrane, while shear forces, derived from a range of physiologicalblood flow values, are continuously applied to the second surface of theporous membrane or to the plated nonparenchymal hepatic cells. The shearforces applied to the second surface of the porous membrane mimic theflow through hepatic sinusoids which occurs in vivo. The shear rate issuitably about 0.1 dynes/cm² to about 3.0 dynes/cm², about 0.2 dynes/cm²to about 2.5 dynes/cm², about 0.3 dynes/cm² to about 1.0 dynes/cm² orabout 0.4 dynes/cm² to about 0.8 dynes/cm². For example, the shear ratecan be about 0.6 dynes/cm². Alternatively, the shear rate can be about2.0 dynes/cm².

In the physiologic in vitro liver model, one or more factors are presentin the culture media. These one or more factors can be added to themedia at concentrations which are capable of maintaining the mimickingof the physiologic liver condition in vitro for a period of time underthe shear force, where the same concentrations of these factors areincapable of maintaining the mimicking of the physiologic livercondition in vitro for the period of time in the absence of the shearforce. For example, the factors may comprise insulin, glucose, or acombination of insulin and glucose. The glucose and insulin are suitablypresent in reduced concentrations as compared to the concentrationswhich are typically used in static cultures (about 17.5 mM glucose andabout 2 μM insulin). For example, the glucose may be present in theculture media at a concentration of about 5 mM to about 10 mM, or at aconcentration of about 5.5 to about 7 mM, e.g., at a concentration ofabout 5.5 mM. The insulin may be present in the culture media at aconcentration of about 0.05 nM to about 5 nM, for example about 0.1 nMto about 3 nM, or about 0.5 to about 2.5 nM, e.g., at a concentration ofabout 2 nM. The one or more factors are suitably added to the culturemedia before or concurrently with application of the shear force.

The concentrations of the one or more factors are suitably capable ofmaintaining the mimicking of the physiologic liver condition in vitrofor at least about 7 days, at least about 14, days, at least about 21days, at least about 30 days, or longer.

Mimicking of the physiologic liver condition can be assessed by a numberof methods. In general, a change in a level of a marker of thephysiologic liver condition in the hepatocytes or nonparenchymal hepaticcells or in the culture media upon application of the shear force, ascompared to the level of the marker in the hepatocytes or nonparenchymalhepatic cells or in the culture media in the absence of application ofthe shear force confirms mimicking of the physiologic liver condition.For example, mimicking of the physiologic liver condition can beassessed by examining the hepatocytes or nonparenchymal hepatic cellsfor the expression of genes or proteins involved in maintaining theliver in a physiologic state (e.g., in hepatocytes, metabolic andinsulin/glucose/lipid pathway genes); examining the hepatocytes forlipid accumulation; examining the hepatocytes or nonparenchymal hepaticcells for changes in differentiated function (e.g., in hepatocytes,measuring urea and albumin secretion); examining the hepatocytes ornonparenchymal hepatic cells for changes in metabolic activity (e.g., inhepatocytes, using cytochrome p450 assays) or transporter activity; orby examining the hepatocytes or nonparenchymal hepatic cells formorphological changes. The physiologic condition of the liver can alsobe assessed by comparing the response of the hepatocytes ornonparenchymal hepatic cells to xenobiotics, nutrients, growth factorsor cytokines to the in vivo liver response to the same xenobiotics,nutrients, growth factors or cytokines.

As described further in Example 12 below, unlike hepatocytes culturedunder static conditions, hepatocytes cultured in the physiologic invitro liver model described in U.S. Patent Application Publication No.2013/0309677 and PCT Publication No. 2013/0158939 maintain theirresponsiveness to glucagon, insulin, and glucose substrates. Thus,responsiveness to glucagon, insulin, or one or more glucose substrates(e.g., using a gluconeogenesis assay) can also be used to assessmimicking of the physiologic liver condition. Suitable glucosesubstrates include glycerol, lactate, pyruvate, or combinations thereof(e.g., a combination of lactate and pyruvate). Moreover, because thehepatocytes maintain responsiveness to glucagon, the physiologic invitro liver model can be used for in vitro testing of drugs thatinteract with the glucagon receptor (e.g., glucagon receptorantagonists).

In addition, hepatocytes cultured in the physiologic in vitro livermodel described in U.S. Patent Application Publication No. 2013/0309677and PCT Publication No. 2013/0158939 display induction and toxicityresponses to drugs at concentrations much closer to in vivo and clinicalC_(max) levels than static culture systems. Thus, this model can be usedfor in vitro testing of drugs and compounds at concentrations within theconcentration range of the drug or compound that achieves an effect invivo.

Fatty Liver

The methods described in U.S. Patent Application Publication No.2013/0309677 and PCT Publication No. 2013/0158939 can also be used tocreate an in vitro model of fatty liver disease. Lipid regulation withinhepatocytes is a complex and dynamic process. Triglyceride buildup canoccur as a consequence of increased fatty acid uptake from a high fatdiet, increased peripheral lipolysis, or from increased de novolipogenesis. Insulin and glucose are key regulators of de novolipogenesis and contribute to increased triglyceride content withinhepatocytes by stimulating triglyceride synthesis as well as inhibitingfatty acid metabolism by beta oxidation.

Non-alcoholic fatty liver disease (NAFLD) is correlated with obesity,type II diabetes, and metabolic syndrome in the presence of insulinresistance. NAFLD is characterized by hepatic steatosis (excessive lipidaccumulation in the liver) that if left untreated progresses toinflammatory changes (steatohepatitis) and cirrhosis. Many animal modelsinduce steatosis through a hyperglycemic-hyperinsulinemic environment(e.g., through use of a low fat/high carbohydrate diet to stimulatelipogenesis). However, current in vitro hepatocyte models lack anadequate insulin-glucose response to induce the same, probably onaccount of the superphysiological levels of insulin/glucose required tomaintain hepatocytes in culture under static conditions. Such in vitromodels fail to induce fatty changes in hepatocytes through insulin andglucose, perhaps due to impaired insulin responsiveness of hepatocytesunder static culture conditions and rapid dedifferentiation of thehepatocytes in vitro.

By contrast, as described above with respect to the physiological livermodel, hepatocytes cultured in the presence of controlled liver-derivedhemodynamics and transport retain differentiated function, morphology,and response at physiological glucose and insulin levels. In thissystem, introducing high concentrations of insulin and glucose (a“disease milieu”) induces fatty changes in the hepatocytes. Thus,controlled hemodynamics and transport produces a more physiologicalresponse to insulin and glucose in the hepatocytes, thereby inducing thefatty changes associated with steatosis in a hyperinsulemic,hyperglycemic environment as is typically seen initially under insulinresistant conditions of diabetes. In addition, hepatocytes cultured inthe presence of controlled hemodynamics and transport display inductionand toxicity responses to drugs at concentrations much closer to in vivoand clinical C_(max) levels than static culture systems. This systemtherefore provides an in vitro model of fatty liver disease.

In this model, the hepatocytes are generally plated in the same manneras described above for the physiological liver model. Hepatocytes areplated on a surface within a cell culture container, and shear forcesare applied indirectly to the plated hepatocytes. For example, thehepatocytes are suitably plated on a first surface of a porous membrane,where the porous membrane is suspended in a cell culture container suchthat the first surface is proximal and in spaced relation to a bottomsurface of the cell culture container, thereby defining within the cellculture container a lower volume comprising the hepatocytes and an uppervolume comprising a second surface of the porous membrane. The shearforce is applied to the second surface of the porous membrane in theupper volume of the container.

At least one layer of one or more extracellular matrix components cansuitably be deposited on the first surface of the porous membrane. Thehepatocytes are then plated on the extracellular matrix component(s).One or more additional layers of the extracellular matrix component(s)can then be deposited on top of the hepatocytes, such that thehepatocytes are substantially surrounded by the extracellular matrixcomponent(s). The extracellular matrix component suitably comprisesheparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid,a collagen, an elastin, a fibronectin, a laminin, a vitronectin, orcombinations thereof. For example, the extracellular matrix componentcan comprise collagen.

One or more additional cell types can be plated on a surface within thecell culture container or suspended in the culture media. For example,nonparenchymal hepatic cells are suitably plated on the second surfaceof the porous membrane, and the shear force is applied to the platednon-parenchymal cells. The nonparenchymal cells may include hepaticstellate cells, sinusoidal endothelial cells, Kupffer cells, orcombinations thereof. The hepatocytes and nonparenchymal hepatic cellsare suitably primary cells isolated from the liver of an animal, forexample from the liver of a human. Alternatively, the hepatocytes and/orthe nonparenchymal hepatic cells are immortalized cells.

Media is suitably continuously perfused on both sides of the porousmembrane, while shear forces, derived from a range of physiologicalblood flow values, are continuously applied to the second surface of theporous membrane or to the plated nonparenchymal hepatic cells. The shearforces applied to the second surface of the porous membrane mimic theflow through hepatic sinusoids which occurs in vivo. The shear rate issuitably about 0.1 dynes/cm² to about 3.0 dynes/cm², about 0.2 dynes/cm²to about 2.5 dynes/cm², about 0.3 dynes/cm² to about 1.0 dynes/cm² orabout 0.4 dynes/cm² to about 0.8 dynes/cm². For example, the shear ratecan be about 0.6 dynes/cm². Alternatively, the shear rate can be about2.0 dynes/cm².

In the in vitro fatty liver model, one or more factors are present inthe culture media. These one or more factors are added to the media atconcentrations which are capable of maintaining the mimicking of fattyliver disease in vitro for a period of time under the shear force, thesame concentration of factor being incapable of maintaining themimicking of fatty liver disease for the period of time in the absenceof the shear force. The factors may comprise, for example, insulin,glucose, or a combination thereof. The glucose is suitably present inthe culture media at a concentration of about 10 mM to about 25 mM,about 12 mM to about 20 mM, or about 14 mM to about 18 mM, e.g., about17.5 mM. The insulin is suitably present in the culture medium at aconcentration of about 1 μM to about 3 μM, about 1.5 μM to about 2.5 μM,or about 1.8 μM to about 2.2 μM, e.g., about 2 μM. The one or morefactors are suitably added to the culture media before or concurrentlywith application of the shear force.

The concentrations of the one or more factors are suitably capable ofmaintaining the mimicking of fatty liver disease condition in vitro forat least about 7 days, at least about 14, days, at least about 21 days,at least about 30 days, or longer.

Mimicking of fatty liver disease can be assessed by a number of methods.In general, a change in a level of a marker of fatty liver disease inthe hepatocytes or nonparenchymal hepatic cells or in the culture mediaupon application of the shear force, as compared to the level of themarker in the hepatocytes or nonparenchymal hepatic cells or in theculture media in the absence of application of the shear force confirmsmimicking of fatty liver disease. For example, mimicking of fatty liverdisease can be assessed by examining the hepatocytes or nonparenchymalhepatic cells for the expression of genes or proteins involved in thefatty liver disease state (e.g., in hepatocytes, metabolic andinsulin/glucose/lipid pathway genes); examining the hepatocytes forlipid accumulation (e.g., in hepatocytes, measuring triglyceride levelsor visualizing lipid droplets); examining the hepatocytes ornonparenchymal hepatic cells for changes in differentiated function(e.g., in hepatocytes, measuring urea and albumin secretion); examiningthe hepatocytes or nonparenchymal hepatic cells for changes in metabolicactivity (e.g., in hepatocytes, using cytochrome p450 assays) ortransporter activity; or by examining the hepatocytes or nonparenchymalhepatic cells for morphological changes. Sequelae to fatty liver changescan also be assessed by measuring the changes in oxidative state of thehepatocytes and the changes in surrounding extracellular matrixcomposition and amount.

The methods described in U.S. Patent Application Publication No.2013/0309677 and PCT Publication No. 2013/0158939 are furtherillustrated by Examples 12-14 below.

DEFINITIONS

For purposes of the inventions described herein, the term “hemodynamic”means blood flow that mimics the blood flow in vivo in a tumor or tissueof interest. For example, when blood flow in the microvasculature of atumor, the acceleration/deceleration rates, flow reversal, forward basalflow, etc. are some parameters characterizing arterial hemodynamic flow.In some tissues, such as the liver, a constant blood flow may be used tocharacterize in vivo hemodynamics.

The term “subject” means an animal (e.g., a genetically modified animalor a human). The animal can include a mouse, rat, rabbit, cat, dog,primate, guinea pig, hamster, monkey, cow, pig, horse, goat, sheep, birdor fish, or any animal typically used in medical research.

Having described the invention in detail, it will be apparent thatmodifications and variations are possible without departing from thescope of the invention defined in the appended claims.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present invention.

Example 1 Plating and Culturing Methods

Porous membranes used for plating were porous membranes of TRANSWELLcell culture inserts (polycarbonate, 10 μm or 18 μm thickness and 0.4 μmpore diameter, 75 mm insert diameter, custom ordered from Corning). Toprepare the inserts for cell plating, both surfaces of the porousmembrane were coated with gelatin (0.14% solution in sterile water). Theporous membranes separate the cell types in the cultures but allow forcell-cell interactions to occur through the pores of the membrane.

The following cell types were used: human fibroblasts (Hs888Lu lungfibroblasts, American Type Culture Collection (ATCC) CCL-211), dtumorcells (A549 human non-small cell carcinoma (NSCLC) tumor cells, ATCCCL-185), and human dermal microvascular endothelial cells (HMVECad,Gibco, catalogue #C-011-5C). Each of these cell lines was maintained,passaged and plated in DMEM base (without phenol red) containing 10%Fetal Bovine Serum (FBS), 100 units/mL penicillin (P), 100 μg/mLstreptomycin (S), 2 mM L-glutamine (L-glut), 1 mM sodium pyruvate (NaP),1× non-essential amino acids (NEAA, HyClone SH30238.011), and 4.5 g/LD-glucose.

The human fibroblasts were plated on the lower surface of the porousmembrane of a cell culture insert at a plating density of 3.0×10³cells/cm² and allowed to adhere for at least two hours in a humidifiedchamber at 37° C. with 5% CO₂.

To generate tri-cultures, a secondary porous membrane was cut from a 75mm insert (TRANSWELL, polycarbonate, 10 μm or 18 μm thickness and 0.4 μmpore diameter, custom ordered from Corning) and prewet with a collagensolution (2.0 mg/mL rat tail collagen in 1×PBS, pH 7.4) by dipping themembrane into a 100 mm dish containing 1.0 mL of collagen solution,wetting both sides of the membrane and allowing any excess collagensolution to drip off the membrane. The secondary membrane was placed ontop of the fibroblasts plated on the lower surface of the porousmembrane of the cell culture insert and allowed to adhere and thecollagen to solidify for one hour in a humidified chamber at 37° C. with5% CO₂. The secondary membrane separating the stromal fibroblasts andthe tumor cells was needed to perform the RNAseq transcriptomicsdescribed hereinbelow, but can be omitted as shown, for example, in theconfigurations depicted in FIGS. 5-8.

Three plating conditions were used: (1) a plating configuration that wassubstantially free of exogenously added ECM (referred to in the Examplesand Figures herein as “no collagen,” “NC,” “no ECM,” or “no matrix”);(2) a plating configuration wherein the tumor cells were plated on alayer of exogenously added collagen (referred to in the Examples andFigures herein as “collagen layer” or “CL”); and (3) a platingconfiguration wherein the tumor cells were plated on a layer ofexogenously added collagen, and wherein an additional layer of collagenwas then added on top of the plated tumor cells such that the collagensubstantially surrounded the tumor cells (referred to in the Examplesand Figures herein as a “collagen sandwich” or CS).

In experiments where the plating configuration was substantially free ofexogenous ECM, tumor cells were plated directly onto the opposingsurface secondary membrane (i.e., the surface not in contact with thefibroblasts). In this configuration, the only exogenous ECM added to thecell culture container was the collagen into which the secondarymembrane was immersed.

In experiments where a collagen layer or collagen sandwich configurationwas desired, 900 μL of collagen solution (2.0 mg/mL rat tail collagen in1×PBS, pH 7.4) was applied evenly to the prewet opposing surface of thesecondary membrane (i.e., the surface not in contact with thefibroblasts) and allowed to adhere and collagen to solidify for one hourin a humidified chamber at 37° C. with 5% CO₂. This generated anapproximately 200 micron thick collagen layer. This concentration ofcollagen produces a gel with a rigidity that mimics the stiffness of thetumor microenvironment.

Human tumor cells were then plated on the collagen layer on the surfaceof the secondary membrane at a plating density 3.0×10³ cells/cm² andallowed to adhere for at least two hours in a humidified chamber at 37°C. with 5% CO₂. After cells had adhered, the cell culture inserts wereinverted and placed into a 100 mm culture dish containing 15 mL DMEMbase (without phenol red)+10% FBS, P/S, L-glut, NaP, NEAA and D-glucose(9 mL of the culture medium was in the lower volume, and 6 mL of theculture medium was in the lower volume).

If a collagen sandwich was desired (i.e., a plating configurationwherein collagen substantially surrounds the cells plated on the lowersurface of the porous membrane of the cell culture insert), anadditional 900 μL of collagen solution (2.0 mg/mL rat tail collagen in1×PBS, pH 7.4) was evenly applied on top of tumor cells and allowed tosolidify for one hour in a humidified chamber at 37° C. with 5% CO₂.This generated an approximately 200 micron thick layer of collagen ontop of the cells plated on the lower surface of the porous membrane ofthe cell culture insert). After this top layer of collagen hadsolidified, the cell culture inserts were inverted and placed into a 100mm culture dish containing 15 mL DMEM base (without phenol red)+10% FBS,P/S, L-glut, NaP, NEAA and D-glucose (9 mL of the culture medium was inthe lower volume, and 6 mL of the culture medium was in the lowervolume).

In all configurations, following plating of the fibroblasts and tumorcells, cells were allowed to grow for 48 hours in a humidified chamberat 37° C. with 5% CO₂. Human endothelial cells were then plated on theupper surface of the porous membrane of the cell culture insert at aplating density of 5.0×10⁴ cells/cm² and allowed to adhere for 24 hoursin a humidified chamber at 37° C. with 5% CO₂ under static conditionsThe resulting tri-culture plating configuration is illustrated in FIG.9.

After endothelial cells had adhered, the tri-cultures were prepared forexperimental hemodynamics and transport. Tri-cultures of tumor cells,fibroblasts, and endothelial cells were either maintained under staticconditions (for static controls) or placed into a cone-and-plate device,the cone was lowered into the upper volume, and the cone was rotated toapply a shear force upon the endothelial cells. In cultures subjected toshear, transport was controlled in the system by perfusing cell culturemedium into and out of both the upper and lower volumes of the cellculture dish via inlets 17 and outlets 19 in the upper and lowervolumes, as depicted in FIG. 9. In experiments wherein drugs were used,drug solutions were added to the upper volume, which represents thevascular compartment.

The correlation of color Doppler ultrasound with histologic specimensfrom both benign and malignant tumors suggests that constant flow ismore representative of the true neovascularization of malignant lungcancers (Hsu et al., 2007; Hsu et al. 1996; Görg et al. 2003). Amonophasic low-impedance waveform more characteristic of peripheralbronchial artery blood flow was selected for application to thetri-cultures. Blood flow in this region is slower and lacks asignificant systolic/diastolic variation, as illustrated in FIG. 4A-C.For contrast, the shear stress pattern from a pulmonary lesion near thepulmonary artery is illustrated in FIG. 4A-C.

Example 2 Morphology of Cells Cultured in In Vitro TumorMicroenvironment Tri-Cultures

Tri-cultures prepared as described above and as illustrated in FIG. 9were fixed after seven days of hemodynamic shear stress in 4%Paraformaldehyde (Electron Microscopy Sciences) for 20 minutes at roomtemperature (RT), washed in phosphate Buffer saline (PBS) with Calciumand Magnesium (Fisher Scientific) and stored at 4° C. until processed.Samples were permeabilized with 0.1% Triton for 20 min, and stained withALEXA FLUOR 488 (a fluorescent dye)-labeled Phalloidin (1:100; LifeTechnologies) to stain for F-actin and TO-PRO-3 nuclear stain (1:2000;Life Technologies) for one hour at room temperature. After three washeswith PBS, samples were mounted between coverslips using FLUOROMOUNT G(an aqueous mounting medium, Southern Biotech). Images were taken with aNikon ECLIPSE Ti Confocal Microscope using 20× oil immersion objectives.

Confocal microscopy images are provided in FIG. 13. FIGS. 13A, 13B, and13C the morphology of the human dermal microvascular endothelial cells(FIG. 13A), the human fibroblasts (FIG. 13B) and the human A549 NSCLCtumor cells (FIG. 13C) plated in the collagen sandwich platingcondition. FIGS. 13D, 13D, and 13F show the morphology of the A549 tumorcells under the three different plating conditions: no ECM (FIG. 13D),collagen layer (FIG. 13E), and collagen sandwich (FIG. 13F). Thephenotype of the A549 cells in the collagen sandwich was spheroidalwhereas in the no ECM condition, the A549 cells pile up on one anotherbut do not form spheres.

Example 3 Tumor Cell Growth in In Vitro Tumor MicroenvironmentTri-Cultures

The growth rate of the human lung carcinoma cell line A549 (ATCC,Manassas, Va.) was determined in multiple matrix conditions when exposedto tumor capillary hemodynamics. Cells were plated as shown in FIG. 9.Hs888Lu human lung fibroblasts were plated onto the porous membrane ofthe cell culture insert at an initial plating density of 3×10³cells/cm². A secondary membrane was applied as described above inExample 1 and A549 tumor cells were then seeded at an initial density of3×10³ cells/cm² in each of the three matrix conditions as describedabove in Example 1: (1) cells plated directly onto the secondarymembrane (no matrix); (2) cells plated onto single layer of collagen(collagen layer); or (3) cells plated in a collagen sandwich. After 48hours of incubation in a static environment, dermal microvascularendothelial cells were plated on the upper surface of the porousmembrane of the cell culture insert at an initial plating density of5×10⁴ cells/cm². Cultures were cultured for up to seven days understatic conditions or subjected to hemodynamic flow and transport. Forstatic cultures, cell number was determined at the time of seeding (Day0) and days 2, 4 and 7 using the CYQUANT Cell Proliferation Assay Kitaccording to the manufacturer's instructions (Molecular Probes, Eugene,Oreg.). For cultures subjected to hemodynamic flow and transport, cellnumber was determined on the day cultures were subjected to hemodynamicflow and transport (Day 0) and on days 2, 4, and 7 of hemodynamic flowand transport. Data are shown in FIGS. 14A and 14C and are presented asthe mean number of cells from duplicate cultures for each of the threematrix conditions. For each condition, the growth rates were compared tothe growth rate of A549 cells grown in plastic two-dimensional (2D)tissue culture dishes without matrix and in the absence of hemodynamicflow and transport.

As shown in FIG. 14A, hemodynamic flow attenuated the growth rate ofA549 tumor cells grown in the tri-cultures in collagen sandwiches ascompared to static 2D culture. There has been a long standing disconnectbetween the doubling time of cells grown in static 2D cultures and thedoubling time of in vivo tumors (Cifone, 1982), be that in xenografttumors derived from cell lines or the rate of tumor growth in patients.When the growth rate of A549 NSCLC cells grown in static 2D cultures andthe tri-cultures subjected to hemodynamic conditions were compared, thetumor cell growth in the tri-cultures subjected to hemodynamicconditions was diminished relative to growth in the static 2D system(FIG. 14A). The growth rate of A549 cells in static 2D conditions was8-fold higher in phase II than in the tri-cultures subjected tohemodynamic conditions. While the growth rate of the A549 cells in thetri-cultures subjected to hemodynamic conditions still exceeds the rateof A549 xenografts (Basu et al., 2011; Chou et al., 2008; Li et al.2013), the growth rate in the tri-cultures subjected to hemodynamicconditions was closer to the physiologic rate than that of static 2Dcultures. 14B. FIG. 14B shows the qualitative differences in the growthrate between A549 cells grown in static 2-dimensional cultures(“static”), the tri-cultures subjected to hemodynamic conditions (“Invitro tumor microenvironment”), and xenografts. The darkly shadedspheres in FIG. 14B represent the starting tumor size, and the lightlyshaded areas and arrows represent tumor growth over time.

FIG. 14C illustrates that the decrease in growth occurred in all threematrix conditions examined. Thus, hemodynamic flow and transport, notextracellular matrix, appear to be the driving contributors effectinggrowth rate.

Example 4 Assessment of Cell Density and Monolayer Integrity ofEndothelial Cells

Endothelial cell density and monolayer integrity of endothelial cellscultured according to the methods described above in Example 1 can beevaluated by fixing and immunostaining the endothelial cells on theporous membrane for the endothelial junction protein VE-cadherin andexamining the monolayer by confocal microscopy.

Example 5 Morphological Assessment of Tumor Cells

To assess the morphology of tumor cells cultured using the methodsdescribed above in Example 1, morphologies of the tumor cells can bedetermined by immunostaining the tumor cells on the porous membrane forE-cadherin and for actin using fluorescently labeled phalloidin. Toquantitate the extension of invasive structures (invadopodia) throughthe pores of the porous membrane, the cultures can be fixed andimmunostained for E-cadherin (for the tumor cells) and VE-cadherin (forendothelial cells), and a cross-section of the porous membrane can beanalyzed by confocal microscopy.

Example 6 Endothelial Cell Monolayer Permeability

In in vivo tumor-vasculature regions, an increase in vessel permeabilityoccurs due to the secretion of numerous growth factors from the tumorcells (Mukaida et al., 2012; Bradford et al., 2013). To demonstrate thattumor cells alter endothelial cell barrier function in the tri-culturesystems described above in Examples 1, permeability assays wereperformed to measure increases in endothelial monolayer permeability. Inthis assay, horseradish peroxidase (HRP) was added to the upper volumeand accumulation of HRP in the presence and absence of tumor cells wasmeasured over time.

After establishing tumor cell/fibroblast/endothelial cell cocultures asdescribed in Example 1 and as illustrated in FIG. 9, a known mass of lowmolecular weight HRP was added to the upper volume and allowed todiffuse. Following a 15-60 minute incubation period, media samples wereremoved from the lower volume. Application of shear stress upon theendothelial cells was continued during this process. HRP that diffusedthrough the endothelial cells, porous membranes, fibroblasts, and A549tumor cells was detected by guaiacol oxidation. Endothelial cellmonocultures (grown on porous membranes without the tumor cells) thatwere subjected to the shear stress were assayed in parallel as acontrol.

The results of this assay are provided in FIG. 15 and demonstrate thatthe NSCLC A549 cells increase endothelial cell permeability. RelativeHRP accumulation in the absence and presence of NSCLC A549 cells isshown. In FIG. 15, “no tumor cells” indicates the endothelial cellmonocultures, and “tumor cells” indicates the tumorcell/fibroblast/endothelial cell cocultures.

Example 7 Transcriptosomic Profile of Tumor Cells in In Vitro TumorMicroenvironment Tri-Cultures

Next-generation RNA sequencing (RNA-seq) was used to compare thetranscriptosome of A549 tumor cells grown under three differentconditions: (1) in static two dimensional cultures; (2) in xenografts inpropagated subcutaneously in athymic nude mice; and (3) in thetri-cultures generated as described above in Example 1 and subjected tohemodynamic conditions. RNA was isolated from tumors cells grown for 2,4, and 7 days under hemodynamic conditions. This comparison indicatedthat transcriptosome of the tri-cultures subjected to hemodynamicconditions more closely resembled the in vivo xenografts than the invitro static 2D cultures.

RNA-seq data were generated using a reverse strand library preparationfrom A549 NSCLC cells grown tri-cultures as described above in Example 1and as illustrated i FIG. 9, under the three different matrixconfigurations. Approximately 20 million 50 bp paired-end reads weresequenced per sample. Raw sequence reads were aligned to the Universityof California Santa Cruz (UCSC) annotations of known isoforms in thehg19 assembly of the human genome using the Bowtie aligner (Langmead etal. 2009). Estimates of read counts per isoform were computed using theeXpress tool (Roberts and Pachter, 2012). Gene-wise counts weregenerated by summing the estimated counts across isoforms for each gene.Genes with low counts across the entire experiment were not consideredin downstream analyses. Specifically, genes that were detected at alevel of at least 2 counts per million in fewer than 5 samples werediscarded. After filtering genes with low signal, library sizes werenormalized using the Trimmed Mean of M-values (TMM) method (Robinson andOshlack, 2010).

To distinguish human from mouse RNA in the xenograft samples, we used astrategy similar to that employed in Raskatov et al., 2012. Raw readswere aligned to the human hg19 and mouse mm10 transcriptomessimultaneously. The alignments were then quantified for each transcript(human and mouse) using eXpress. All eXpress estimates computed formouse transcripts were discarded, and estimates for human transcriptswere used in downstream analyses.

Gene-wise differential expression analysis was performed on the filteredand TMM-normalized abundance estimates using edgeR (Robinson et al.,2010). The NSCLC gene set is from the Kyoto Encyclopedia of Genes andGenomes (KEGG) database (Ogata et al., 1999). The gene set consists of:AKT3, CDK4, CDK6, RASSF1, E2F1, E2F2, E2F3, EGF, EGFR, ERBB2, AKT1,AKT2, EML4, GRB2, HRAS, ARAF, KRAS, NRAS, PDPK1, PIK3CA, PIK3CB, PIK3CD,PIK3R1, PIK3R2, PLCG1, PLCG2, PRKCA, PRKCG, MAPK1, MAPK3, MAP2K1,MAP2K2, RAF1, RARB, CCND1, RXRA, RXRB, SOS1, SOS2, BRAF, TGFA, RASSF5,PIK3R3, FOXO3, BAD, CASP9, RB1, STK4.

The results of RNA-seq transcriptosomal profiling are shown in FIGS.16-18. FIG. 16 provides a dendogram (FIG. 16A) and a heatmap (FIG. 16B)showing the clustering of samples based on the gene-centered log 2expression values of all 14,159 genes that reach the signal threshold.A549 cells grown in the tri-cultures generated as described above andsubjected to hemodynamic conditions clustered based on the matrixconditions. “CL”=collagen layer; “CS”=collagen sandwich; NC=no collagen.Xenograft samples clustered separately, with the exception of the nocollagen condition on day 7, suggesting that increased time in thedevice in the absence of collagen more faithfully recapitulates thetranscriptomic profile of xenograft tissue, relative to the otherculture conditions. The hierarchical clustering was performed using anaverage linkage criteria, and Spearman rank correlation as the distancemetric.

FIG. 17 provides a dendogram (FIG. 17A) and a heatmap (FIG. 17B) showingthe clustering of samples based on the log 2 fold changes of the 7935genes differentially expressed between the xenograft and plasticconditions at a 5% false discovery rate (FDR). Xenograft samples clusterwith the samples from tri-cultures subjected to hemodynamic conditionsthat do not contain exogenous collagen. This indicates that thetranscriptomic differences between xenograft and static two-dimensionalcultures are better reproduced in the absence of exogenous collagen. Thehierarchical clustering was performed using an average linkage criteria,and Spearman rank correlation as the distance metric. “CS”=collagensandwich; “NC”=no collagen; “plastic”=static 2D cultures.

FIG. 18 provides a dendogram (FIG. 18A) and a heatmap (FIG. 18B) showingthe clustering of samples based on the log 2 fold changes (conditionversus static 2D cultures (“plastic”)) of the 48 genes in this datasetthat are annotated with “Non-small cell lung cancer” in the KEGGdatabase. Xenograft samples cluster with the samples from tri-culturessubjected to hemodynamic conditions that do not contain collagen. Thisindicates that the transcriptional responses of genes that are highlyrelevant to the disease phenotype are more similar to xenografts in theabsence of collagen than in the presence of collagen. The hierarchicalclustering was performed using an average linkage criteria, and Spearmanrank correlation as the distance metric. CS=collagen sandwich; NC=nocollagen; “plastic”=static 2D cultures. The 48 genes in the data set,the expression of which is indicated in the rows of the heatmap, fromtop the top row to the bottom row are: EGF, RXRB, CASP9, CDK4, RASSF5,PRKCA, FOXO3, RARB, PIK3R2, MAPK3, RXRA, AKT2, GRB2, ARAF, MAP2K2, AKT1,RASSF1, HRAS, BRAF, EML4, STK4, NRAS, MAPK1, RAF1, MAP2K1, PDPK1,PIK3CB, TGFA, BAD, PLCG2, E2F2, E2F1, PRKCG, EGFR, CDK6, PLCG1, ERBB2,PIK3R3, SOS2, PIK3R1, KRAS, SOS1, PIK3CA, CCND1, PIK3CD, E2F3, AKT3, andRB1.

Example 8 Molecular Activity Profiling of Endothelial Cells and TumorCells

In in vivo tumor-vasculature regions, tumor cell proliferation andinvasion is modulated by endothelial cells in close proximity to thetumor cells. Qualitative and quantitative gene expression and cytokinesecretion profiles for both the endothelial cells and the tumor cellscultured according to the methods described in Example 1 can begenerated. Such assays are used to monitor the expression of angiogenicand tumorigenic factors and demonstrate that endothelial cells culturedunder tumor-derived hemodynamic flow alter the molecular signature ofthe tumor cells, e.g., by enhancing markers of tumor molecular activityas measured using next generation mRNA sequencing and cytokine andgrowth factor secretion profiling.

After establishing tri-cultures as described in Example 1 above, mRNAcan be collected from endothelial and tumor cells for analysis. Table 1lists genes in both endothelial and tumor cells that are known to beregulated in the tumor microenvironment in vivo. This panel of genesserves as an initial molecular assessment of the tri-cultures describedin Example 1 to demonstrate that the heterotypic cell-cell communicationand hemodynamic flow and transport in the coculture systems affect themolecular activity of the tumor microenvironment. Table 1 (right) alsolists a series cytokine and growth factors that are profiled using amultiplexing platform (MAGPIX), which is capable of performingqualitative and quantitative analysis of angiogenic factors such as VEGFand angiopoietins. Because of the design of the system, media can becollected in real-time from the endothelial cell and tumor cells layersseparately for analysis.

TABLE 1 mRNA MAGPIX Gene Function EC A549 EC 549 VEGFA Angiogenesis ✓ ✓✓ ✓ VEGFC Angiogenesis ✓ ✓ ✓ ✓ VEGFD Angiogenesis ✓ ✓ ✓ ✓ ANG1Angiogenesis ✓ ✓ ANG2 Angiogenesis ✓ ✓ ✓ ✓ FGF-2 Angiogenesis ✓ ✓ PLGFAngiogenesis ✓ ✓ EGF Proliferation ✓ ✓ ✓ ✓ EGFR Proliferation ✓ MKI67Proliferation ✓ ✓ PCNA Proliferation ✓ ✓ VIM Invasion ✓ CDH1 Invasion ✓CDH2 Invasion ✓ IL-6 Inflammation ✓ ✓ IL-8 Inflammation ✓ ✓ ✓ ✓ NF-kBInflammation ✓ eNOS Inflammation ✓ KLF2 Inflammation ✓ MCP-1Inflammation ✓ ✓ ✓ ✓

After reproducing the baseline molecular activity with a minimum of fivebiological replicates, next-generation sequencing-based mRNAtranscriptomics (RNAseq) can be performed on the endothelial cells andtumor cells cultured as described above in Example 1, and compared tothe following controls: (1) a static co-culture system, (2) endothelialcells subjected to tumor-derived shear stress in the absence of tumorcells, (3) tumor cells in monoculture; and (4) in vivo tumors. TheOncoMine Research database is used to compare and contrast these resultsagainst >73,000 cancer expression profiles obtained from a large varietyof human tumor samples, including clinical outcomes in >27,000 samples,pathway/drug responses in >7,800 samples and >11,000 samples from theCancer Genome Atlas (TCGA). This provides an unbiased assessment of thecoculture tumor microenvironment system.

Example 9 Testing of Anti-Cancer Drugs in In Vitro TumorMicroenvironment Tri-Cultures

The effects of anti-cancer drugs on the A549 tumor cells grown intri-cultures generated as described above in Example 1 and depicted inFIG. 9 were assessed. The following drugs were selected: cisplatin, afront line chemotherapeutic for NSCLC (Rossi et al., 2012; NationalCancer Institute, Non-Small Cell Lung Cancer Treatment (PDQ®)), and twoexperimental small molecule allosteric inhibitors of MEK(AZD6244/Selumetinib) and AKT (MK-2206), which are in clinical trialsfor NSCLC and other cancers (Leijen et al., 2011; National CancerInstitute, Randomized Phase II Study of ADZ6244; Yap et al., 2011;National Cancer Institute, MK2206 and Erlotinib Hydrochloride inTreating Patients with Advanced Non-Small Cell Lung Cancer). For each ofthe three drugs tested in the tri-cultures subjected to hemodynamicconditions, growth inhibition occurred at the clinically relevant humanpatient C_(max).

The IC₅₀s of these three drugs for A549 tumor cells grown under static2D conditions does not approximate the clinically relevant doses used incancer patients. For cisplatin the in vivo C_(max) is 3 μM and thusrepresents the clinical dose that patients with NSCLC receive (Urien etal., 2005). However, the cisplatin IC₅₀ for A549 cells in static 2Dcultures from published results ranges from 6 μM to over 60 μM (Andrianiet al., 2006; Zhang et al., 2013; Zhang et al., 2003; Barr et al.,2013). Thus, cisplatin is routinely in vitro used at 2× to 20× higherconcentrations than what is achievable in vivo for NSCLC patients.Similarly, the A549 IC₅₀ for AZD6244/Selumetinib and MK2206 in static 2Dcultures is significantly higher than the dose achieved in patients (Yehet al., 2007; Meng et al., 2010). For AZD6244/Selumetinib the C_(max) is1.4 μM and the static 2D IC₅₀ is 5 μM, a 3.5-fold increase. For MK2206the C_(max) is 160 nM and the static 2D IC50 is 3 μM, nearly a 19-foldincrease. These differences in C_(max) and static 2D IC₅₀s helpillustrate a major barrier that confronts work in static 2D conditions;non-physiologic drug concentrations are frequently necessary forbiologic effects.

Table 2 summarizes the human and mouse in vitro C_(max)s and the invitro IC50s for static cultures for cisplatin, MK2206, and AZD6422, aswell the concentrations of the drugs tested tri-cultures subjected tohemodynamic conditions.

TABLE 2 Cisplatin MK2206 AZD6422 Human C_(max) 3 μM 160 nM 1.4 μM MouseC_(max) >10 μM 540 nM 5.5 μM Static 2D IC₅₀ >6 μM 3 μM 5 μMConcentration applied to 3 μM 160 nM 1.4 μM tri-cultures subjected tohemodynamic conditions

The IC₅₀ concentrations of cisplatin, MK2206 and AZD6244/selumetinib forthe A549 cell line listed in Table 2 were taken from the literature asdescribed above and estimated to be >6 μM, 3 μM and 5 μM, respectively.Maximal plasma concentrations (C_(max)) in mice were estimated usingpharmacokinetic data from taken from efficacy studies in thepeer-reviewed literature. For cisplatin, the steady state plasma C_(max)was determined to be >10 μM (Johnsson et al., 1995). For MK2206, thesteady state plasma C_(max) was determined to be 540 nM (Piovan et al.,2013). For AZD6244/selumetinib, the steady state plasma C_(max) wasdetermined to be 5.5 μM (Denton and Gustafson, 2011). Maximal plasmaconcentrations (C_(max)) in humans were estimated with pharmacokineticdata from clinical trials using established therapeutic dosingparadigms. For cisplatin, the steady state plasma C_(max) was determinedto be 3 μM (Salas et al., 2006; Urien et al., 2005). For MK2206, thesteady state plasma C_(max) was determined to be 160 nM (Hudis et al.,2013; Yap et al., 2011). For AZD6244/selumetinib, the steady stateplasma C_(max) was determined to be 1.4 μM (Adjei et al., 2011; O'Neilet al., 2011).

The growth rate of the human lung carcinoma cell line A549 wasdetermined in response to vehicle control (“Veh”), cisplatin, MK-2206,and AZD6244 using the human C_(max) doses described above. Tri-cultureswere prepared as described above in Example 1 and as depicted in FIG. 9in the absence of exogenously added ECM and subjected to tumor capillaryhemodynamics. The vehicle control or drug was added to the inflow mediafor the endothelial cell layer and perfused into the upper volume.Tri-cultures were maintained in the presence of absence of drugs for upto seven days under hemodynamic flow and transport. Cell number wasdetermined at day seven using the QUANT-IT PICOGREEN dsDNA Assay Kitaccording to the manufacturer's instructions (Life Technologies, GrandIsland, N.Y.). Data are presented in FIGS. 19A and 19B as the meannumber of cells from duplicate.

FIG. 19A shows the cell number for A549 tumor cells treated with thevehicle control (“Veh”), cisplatin, MK2206, or AZD6422. FIG. 19B showsthe relative cell growth of A549 tumor cells treated with the vehiclecontrol (“Veh”), cisplatin, MK2206, or AZD6422. Cisplatin inhibited A549growth by 48%, MK2206 by 44%, and AZD6422/Selumetinib by 78%. These dataindicate that tumor cells grown in the tri-cultures subjected tohemodynamic conditions respond to both established chemotherapeutics(cisplatin) and experimental small molecule inhibitors (MK2206, AZD6442)at therapeutically relevant concentrations.

Collectively, these data indicate that tumor cells grown in thetri-cultures and subjected to hemodynamic conditions respond to bothestablished chemotherapeutics and experimental small molecule inhibitorsat human patient physiologic doses. It is important to note that asshown in Table 2, the mouse C_(max) at therapeutic doses for cisplatin,AZD6244/Selumetinib and MK2206 is higher than the human patient Cmax. Inmice the C_(max) of AZD6244/Selumetinib is 5.5 μM (Meng et al., 2010,Chung et al., 2009), which is equivalent to the static 2D IC₅₀. ForMK2206 the mouse Cmax is 540 nM (Meng et al., 2010), which is 5-foldhigher than the human patient C_(max). Cisplatin has a mouse C_(max)ranging from 10-100 μM (Andriani et al., 2006; Zhang et al., 2003; Bainet al., 2007), which exceeds the human patient C_(max) and is equivalentto or higher than the static 2D IC₅₀. Thus, the tri-cultures subjectedto hemodynamic conditions may be superior to mouse xenograft studies fortesting experimental drugs at physiologic doses.

Example 10 Further Testing of Anti-Cancer Drugs in In Vitro TumorMicroenvironment Tri-Cultures

Additional anti-cancer drugs can be tested in for their effects on thetumor cells, stromal fibroblasts, and/or microvascular endothelial cellsgrown in tri-cultures generated as described above in Example 1. Theanti-cancer drug can be introduced into the tri-cultures atconcentrations in the culture medium that are within the concentrationrange of the in vivo therapeutic C_(max) in a human. For example, theantiproliferative chemotherapeutic drug carboplatin (Sigma-Aldrich), andthe EGFR inhibitor erlotinib (Cayman Chemical) can be tested. These twodrugs are commonly used in the treatment of lung cancer, theirpharmacokinetics have been well-characterized through clinical trials,and they both have been shown to improve the survival of lung cancerpatients. Carboplatin is a traditional chemotherapeutic drug thatindiscriminately kills rapidly dividing cells, whereas erlotinib is atargeted therapy that is not as broadly toxic.

The drug can be added to the inflow media for the endothelial cell layerand perfused into the upper volume. Initially, the drugs can be appliedto the endothelial cells at two doses, with the highest dose being thein vivo C_(max) level achieved in humans (for carboplatin, 37 μg/ml; forerlotinib, 1 μg/ml), and a 10-fold lower dose, mimicking the lowerdegree of drug penetration observed in many solid tumors in vivo. Abroader range of doses can then used to generate response profiles foreach drug. As a two-dimensional control, A549 cells can be plated ontissue culture-treated dishes and cultured in conditioned media frommicrovascular endothelial cells in the presence of each drug, and cellcounts can be performed after 3 days. As a three-dimensional control,A549 cells can be seeded in a three-dimensional collagen matrix on thebottom of a well of a 6-well dish, and endothelial cells plated on theupper surface of the porous membrane of a cell culture insert insertedinto the well, similar to current in vitro three-dimensional tumormodels. The endpoints described in the Examples above can be used toassess the effect of the anticancer agent, e.g. cell viability, tumorcell invasion, endothelial cell permeability, RT-PCR, mRNA sequencing,and cytokine and growth factor profiling.

Additional anti-cancer drugs can be tested in a similar manner and theirdose-response curves compared with the dose-response curves for the drugin in vivo models. Such drugs include, for example, angiogenesisinhibitors such as the VEGF inhibitor bevacizumab and the VEGFRinhibitor sorafenib.

Example 11 A Model for Tumor Metastasis to the Liver

The metastasis of tumor cells through the vasculature to distal organsis the major cause of mortality in cancer. The liver is a common sitefor tumor metastases because if its rich blood supply. Human tumor cells(e.g., the A549 tumor cells or pancreatic tumor cells) are culturedaccording to the methods described above in Examples 1, under static 2Dconditions, or as xeongrafts. The tumor cells are then extracted andadded to an in vitro liver model system, for example, an in vitro systemliver model system as described in U.S. Patent Publication No. US2013/0309677 and PCT Publication No. 2013/0158939, the contents of bothof which are hereby incorporated by reference in their entirety. Forexample, in an in vitro liver model system described in U.S. PatentApplication Publication No. US 2013/0309677 and PCT Publication No.2013/0158939, hepatocytes are sandwiched in a collagen gel and plated ona first surface of a porous membrane. Sinusoidal endothelial cells areoptionally plated on the second surface of the porous membrane.Additional non-parenchymal cells (e.g., hepatic stellate cells, Kupffercells, or a combination thereof) are optionally plated on the first orsecond surface of the porous membrane. A shear stress that mimics invivo blood flow in the liver is then applied to the non-parenchymalcells on the second side of the porous membrane, and cell culture mediumis perfused into and out of the upper and lower volumes. FIG. 11provides a schematic drawing of such a system.

Tumor cells are extracted from the tri-cultures, xenografts, or 2Dstatic cultures and added to this in vitro liver system. The tumor cellsare either introduced directly into the lower volume containing thehepatocytes, or to the upper volume optionally containing the sinusoidalendothelial cells and/or other non-parenchymal cells. In the lattercase, transmigration of the tumor cells into the lower volume containingthe hepatocytes is assessed. Growth of the tumor cells in the in vitroliver system is also assessed.

Alternatively, a coculture system containing tumor cells, stromalfibroblasts, and microvascular endothelial cells as described above inExample 1 is linked to an in vitro liver model system by tubing that isused to transfer culture medium from the lower or upper volume of thetumor model coculture to the lower or upper volume of the in vitro livermodel system as shown in FIGS. 12A-D. This creates a model of tumormetastasis mimicking the seeding of distal organs by tumor cells invivo.

Efficacy of anticancer agents can be assessed by measuring the growth ofthese in vitro metastases in the in vitro liver model in the presence ofan anticancer agent, as compared to the growth of the in vitrometastases in the absence of the anticancer agent.

Example 12 A Physiologic In Vitro Liver Model

Static hepatocyte cell culturing methods are associated with poor invitro to in vivo correlations, due in part to the absence ofphysiological parameters which maintain metabolic phenotype over time invivo. Restoring physiological hemodynamics and transport retainshepatocyte phenotype and function in vitro compared to the standardstatic hepatocyte collagen gel configuration.

To recreate a cellular hepatocyte system with fluid dynamics andtransport analogous to in vivo liver circulation, a cone-and-platedevice-based technology was employed that has been extensively used tore-establish in vivo blood vessel cell phenotypes by recreating theexposure of vascular endothelial cells to human-derived hemodynamicblood flow forces in vitro. This technology is described in U.S. Pat.No. 7,811,782. The technology was adapted and modified to design a ratliver monoculture system which applies hemodynamic flow and transportconditions reflective of in vivo hepatic circulatory values. Theconfiguration of cells in the device is based on in vivomicroarchitecture of hepatic lobules where cords of hepatocytes areseparated from sinusoidal blood flow by a filtering layer of endothelialcells. This design uses a porous polycarbonate membrane suspended in acell culture container, with primary rat hepatocytes sandwiched in acollagen gel on one side of the porous membrane. The porous membraneacts analogously to the filtering layer of sinusoidal endothelial cellswhich is present in the liver. Media is continuously perfused on bothsides of the porous membrane, while hemodynamic forces, derived from arange of physiological blood flow values, are continuously applied tothe non-cellular side of the porous membrane. The entire set up ishoused in a controlled environment with 5% CO₂ and at 37° C. Aflow-based culture system was effectively created whereby hepatocytesare shielded from direct effects of flow, as they would be in vivo.Recapitulating the hemodynamics and in a system designed to be analogousto the microstructure of the hepatic sinusoid results in stableretention of a differentiated hepatic and metabolic phenotype similar tothat of in vivo liver.

Methods

(i) Animal Surgery and Hepatocyte Isolation

All animals used for the experiments were treated according to protocolsapproved by HemoShear's Animal Care & Use Committee. Hepatocytes wereisolated from male Fischer rats (250 g-350 g) by a modification ofSeglen's two-step collagenase perfusion procedure using a 20 mL/min flowrate (Seglen, Hepatocyte Suspensions and Cultures as Tools inExperimental Carcinogegnesis, J. Toxicology & Environmental Health,5(2-3): 551-560 (1979), the contents of which are hereby incorporated byreference). Briefly, the rats were anaesthetized with isoflurane,following which the abdominal cavity was incised and the inferior venacava was canulated while making an excision was made in the portal veinfor outflow. The liver was perfused in two steps, first with a Ca⁺⁺-freebuffer to flush out blood and break up intercellular junctions, followedby collagenase in a Ca⁺⁺-containing buffer to digest the extracellularcollagen matrix. After the liver was suitably perfused it was excisedand freed of the capsule in a Petri dish under a sterile hood. Anenriched hepatocyte population (˜95% purity) was obtained by twosequential 65 g centrifugation and washing cycles of 10 minutes eachfollowed by a 10 minute spin with 90% PERCOLL (colloidal silicaparticles of 15-30 nm diameter (23% w/w in water) coated withpolyvinylpyrrolidone (PVP); used to establish density gradients that canbe used to isolate cells). The viability of hepatocytes was determinedby trypan blue exclusion test and cells with a viability over 85% areused.

(ii) Cell Culture and Device Operating Conditions

Hepatocyte Culture Media:

For the data shown in FIGS. 20-24, the rat hepatocyte culture mediacontained base media of DMEM/F12 containing high glucose (17.5 mM),supplemented by fetal bovine serum (10% at the time of plating andreduced to 2% for maintenance after 24 hours). The media also containedgentamycin (50 μg/ml), ITS (insulin concentration 2 μMol), 1% NEAA, 1%GLUTAMAX, and dexamethasone (1 μM at plating and 250 nM for maintenanceafter 24 hours).

For the data shown in Table 6 and FIGS. 38 and 39, the rat hepatocyteculture media contained base media of DMEM/F12 containing low glucose(5.5 mM), supplemented by HEPES (3% vol/vol) and fetal bovine serum (10%vol/vol at the time of plating and reduced to 2% for maintenance after24 hours). The media also contained gentamycin (50 mg/ml), ITS (insulinconcentration 2 nMol), 1% NEAA, 1% GLUTAMAX, and dexamethasone (1 μM atplating and 100 nM for maintenance after 24 hours).

To culture human or dog hepatocytes, the culture media contained basemedia of DMEM/F12 containing low glucose (5.5 mM), supplemented by HEPES(3% vol/vol) and fetal bovine serum (10% vol/vol at the time of platingand reduced to 2% for maintenance after 24 hours). The media alsocontained gentamycin (50 mg/ml), ITS (insulin concentration 2 nMol), anddexamethasone (1 μM at plating and 100 nM for maintenance after 24hours).

Collagen Coating and Plating:

Collagen solution was made by mixing Type I Rat Tail Collagen in steriledistilled water, 10λ phosphate buffered saline (PBS) and 0.2N sodiumhydroxide in a predefined ratio (To make up 1 ml, the components were440 μl, 375 μl, 100 μl and 85 μl respectively).

For cultures to be subjected to static conditions, 100 mm tissueculture-treated sterile cell culture dishes were coated with 7 μl/cm² ofcollagen solution. For cultures to be subjected to controlledhemodynamics, the lower surface of the porous membrane of 75 mmTRANSWELLS (polycarbonate, 10 μm thickness and 0.4 μm pore diameter, no.3419, Corning) were coated with 7 μl/cm² of collagen solution. Afterallowing an hour for the solution to gel, the surfaces were washed withDPBS, hepatocytes were plated at a seeding density of 125,000 viablecells/cm², and a second layer of collagen gel added after 4 hours. After1 hour, the TRANSWELLS were inverted and placed into cell culturedishes, and media was added (9 ml in the lower volume and 6 ml in theupper volume). 7 ml of media was added to the tissue culture dishes tobe used for static cultures. After 24 hours, the media was switched tomaintenance media (containing 2% FBS), and the cell culture dishescontaining TRANSWELLS were placed into the cone-and-plate device.Controlled hemodynamics were applied to the surface of the porousmembrane of the TRANSWELL in the upper volume.

Cryopreserved human hepatocytes were procured from commercial vendors(Kaly-Cell, France) and thawed as per the vendor's prescribed protocols.For plating human hepatocytes, a similar procedure to that describedabove for rat hepatocytes was followed, a limited cell-seeding area wasused. The second layer of collagen was applied as described above.

Freshly isolated canine hepatocytes from beagle dogs were procured fromcommercial vendors (Triangle Research Laboratories, Research TrianglePark, N.C.) and processed as per the vendor's prescribed protocols. Forplating canine hepatocytes, a similar procedure to that described abovefor rat hepatocytes was followed, but using a limited cell-seeding area.The second layer of collagen was applied as described above.

Operating Conditions:

The shear stress in dynes/cm² (τ) was calculated for a typical hepaticsinusoid based on the formula for pressure driven flow of a Newtonianfluid through a cylinder,

$\tau = \frac{\Delta\;{P \cdot r}}{2l}$using reference values for pressure gradient across the sinusoid (ΔP),radius of sinusoids (r) and length of the sinusoids (l) from theliterature. As part of an initial optimization process, a range ofapplied shear stress conditions obtained by altering media viscosity andcone speed that resulted in rates within an order of magnitude of thevalue predicted from literature were seen to be correlated withdifferent transport profiles of horse radish peroxidase dye across themembrane. These were tested for gene expression profiles of thehepatocytes 7 days into culture (data not shown). No differences wereobserved between static cultures and those that were simply perfusedwithout any applied shear and based on the gene expression profiles, anoperational shear rate of 0.6 dynes/cm² was selected for all theexperiments described in this Example.

(iii) Assessment of Phenotypic, Functional, Metabolic, and ToxicParameters

RT-PCR:

Changes in metabolic, toxic, and insulin/glucose/lipid pathway geneswere assessed by extracting RNA from hepatocytes from devices run underhealthy and steatotic conditions at the end of the culture period (7 or14 days) and performing RT-PCR on this RNA. The TRANSWELLS were removedfrom the devices and washed with PBS prior to scraping the cells off theporous membrane. Total RNA was isolated using a PURELINK RNA Mini Kit (akit for purification of total RNA from cells) and reverse transcribed tocDNA using the ISCRIPT cDNA Synthesis Kit (a cDNA synthesis kit).Primers were designed for the metabolic genes CYP1A1, CYP1A2, CYP3A2,MDR, and GST as well as the insulin/glucose/lipid pathway genes GPAT,ACC1, IRS-2, PPAR-γ, SREBP, ChREBP, LXR, SCD1, CPT1. Primer sequencesare shown below in Table 3:

TABLE 3 Rat Primer Sequences Gene Forward (SEQ ID NO.)Reverse (SEQ ID NO.) CYP1A1 GCTGCTCTTGGCCGTCACCA (1)TGAAGGGCAAGCCCCAGGGT (2) CYP1A2 CCTGCGCTACCTGCCCAACC (3)GGGCGCCTGTGATGTCCTGG (4) CYP3A2 CGGCGGGATTTTGGCCCAGT (5)CAGGCTTGCCTGTCTCCGCC (6) MDR GCTGCTGGGAACTCTGGCGG (7)CCGGCACCAATGCCCGTGTA (8) GST (Pi CGCAGCAGCTATGCCACCGT (9)CTTCCAGCTCTGGCCCTGGTC (10) subunit)  GPAT AGCGTTGCTCCATGGGCATATAGT (11)TGTCAGGGATGGTGTTGGATGACA (12) ACC1 TGTCATGGTTACACCCGAAGACCT (13)TTGTTGTTGTTTGCTCCTCCAGGC (14) IRS-2 GCGAGCTCTATGGGTATATG (15)AGTCCTCTTCCTCAGTCCTC (16) PPAR-g ATATCTCCCTTTTTGTGGCTGCTA (17)TCCGACTCCGTCTTCTYGATGA (18) SREBP GGAGCCATGGATTGCACATT (19)AGGCCAGGGAAGTCACTGTCT (20) ChREBP CTATGTCCGGACCCGCACGC (21)CTATGTCCGGACCCGCACGC (22) LXR ACTCTGCAACGGAGTTGTGGAAGA (23)TCGGATGACTCCAACCCTATCCTT (24) SCD1 TGTGGAGCCACAGGACTTACAA (25)AGCCAACCCACGTGAGAGAAGAAA (26) CPT1 ATGTGGACCTGCATTCCTTCCCAT (27)TTGCCCATGTCCTTGTAATGTGCG (28) CYP2B1 GAGGAGTGTGGAAGAACGGATTC (29) AGGAACTGGCGGTCTGTGTAG (30) CYP2B2 TCATCGACACTTACCTTCTGC (31)AGTGTATGGCATTTTGGTACGA (32) SORD TCTGTGGCTCGGATGTTCACTACT (33)CGGCCGATCTTGCAGAATTCATCT (34) GSR GGACTATGACAACATCCCTACC (35)CCAACCACCTTCTCCTCTTT (36) APEX1 GCCTAAGGGCTTTCGTTACA (37)ATCCACATTCCAGGAGCATATC (38) MRP3 AGGCCAGCAGGGAGTTCT (39)AGCTCGGCTCCAAGTTCTG (40) MRP4 CAACTCCTCTCCAAGGTGCT (41)ATCTGCTCACGCGTGTTCTT (42)

RNA expression was analyzed by real-time RT-PCR using IQ SYBR GreenSupermix (a PCR reagent mixture for RT-PCR) and a CFX96 Real-Time Systemwith C1000 Thermal Cycler (an RT-PCR detection system and thermalcycler). RNA data were normalized to endogenous expression ofβ-microglobulin and reported as a relative quantity compared to healthycultures.

Human genes assessed for metabolism and toxicity experiments includedCYP1A1. CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP3A4, CYP3A5, GSTA1, UGT1A1,GSR, SORD, TXNRD1, and APEX1. The primer sequences for these are shownin Table 4. Canine genes assessed for metabolism included CYP1A1 andCYP3A12 (primer sequences shown in Table 5).

TABLE 4 Human Primer Sequences Gene Forward (SEQ ID NO.)Reverse (SEQ ID NO.) CYP1A1 GGACCTGAATGAGAAGTTCTACAGC (43)AGCTCCAAAGAGGTCCAAGACGAT (44) CYP2A6 TCATAGCCAAGAAGGTGGAGCACA (45) CCCAATGAAGAGGTTCAACGTGGT (46) CYP2B6 GGGCACACAGGCAAGTTTACAA (47)AGAGCGTGTTGAGGTTGAGGTTCT (48) CYP2C9 TGACTTGTTTGGAGCTGGGACAGA (49) ACAGCATCTGTGTAGGGCATGT (50) CYP2D6 ACGACACTCATCACCAACCTGTCA (51) AGGTGAAGAAGAGGAAGAGCTCCA (52) CYP3A4 CTGCATTGGCATGAGGTTTGCTCT (53)AAATTCAGGCTCCACTTACGGTGC (54) CYP3A5 CTGCATTGGCATGAGGTTTGCTCT (55)AGGGTTCCATCTCTTGAATCCACC (56) GSTA1 GATGCCAAGCTTGCCTTGAT (57)AGGGAAGCTGGAGATAAAGACTGGA (58) UGT1A1 GGCCCATCATGCCCAATATGGTTT (59) GCATCAGCAATTGCCATAGCTTTC (60) SORD TAGCGCCACCAGAAGCGACCAAA (61)TCATTTGGGCCTGGTTCAGGGATA (62) APEX1 CCAGCCCTGTATGAGGACC (63)GGAGCTGACCAGTATTGATGAGA (64) GSR CACTTGCGTGAATGTTGGATG (65)TGGGATCACTCGTGAAGGCT (66) TXNRD1 ATATGGCAAGAAGGTGATGGTCC (67)GGGCTTGTCCTAACAAAGCTG (68)

TABLE 5 Canine Primer Sequences Gene Forward (SEQ ID NO.)Reverse (SEQ ID NO.) CYP1A1 CACCATCCCCCACAGCACAACAAA (69)GCTCTGGCCGGAATGCAAATGGAT (70) CYP3A12 GAGAGAATGAAGGAAAGTCGCC (71)GCCACCAGCTCCAAATCAGA (72) B2MG TCCTCATCCTCCTCGCT (73)TTCTCTGCTGGGTGTCG (74)

Urea and Albumin Assays:

Media collected from static cultures and devices at various time pointswas assayed for albumin using a rat-specific ELISA based kit (BethylLaboratories) as per the manufacturer's protocols. Urea was estimatedfrom the media samples using a standard colorimetric assay (QUANTICHROMUrea Assay Kit, DIUR-500, Gentaur). All measurements between the systemswere normalized to a per million cells/day rate for comparison based onthe volume of media perfused and the number of initially plated cells.

Western Blots:

Following application of controlled hemodynamics, ⅓ of the platedsurface of the porous membrane of the TRANSWELL (˜1.8 million cells) washarvested for protein in 150 μl 1×RIPA buffer containing fresh 150 mMDTT and protease inhibitors (HALT Protease Inhibitor Cocktail (Pierce)+1mM PMSF+200 mM DTT). Samples were sonicated on ice with 5×1 secondpulses, allowed to sit on ice for 30 minutes and centrifuged at 17,000×gfor 10 minutes in a chilled microcentrifuge. Protein determination wasdone using A660 nm Protein Reagent (Pierce). Samples were boiled 70° C.for 10 minutes and then run on a 7.5% TGX gel (a pre-cast polyacrylamidegel, BioRad) before wet-transferring to 0.2 μm PVDF membrane andblocking in 5% non-fat milk at room temperature for 10 minutes.Membranes were incubated overnight at 4° C. in rabbit anti UGT antibody(Cell Signaling, 1:500 dilution). Secondary antibody (Santa Cruz, Goatanti Rabbit HRP, 1:5000 dilution) incubation was at room temperature forone hour. Chemiluminescent signal was developed using SUPERSIGNAL WESTPICO (a chemiluminescent substrate for horseradish peroxidase, Pierce)reagent and captured using an Innotech ALPHAEASE imaging system. Fornormalization, gels were probed for mouse anti β-Actin (Sigma A1978,1:2000 dilution) followed by secondary goat anti mouse HRP (Santa Cruzsc-2005, 1:10,000 dilution).

Immunostaining and Biliary Activity Stain:

Antibodies used: Hnf4a (Santa Cruz sc-8987), E-cadherin (Santa Cruzsc-71009), and anti-MRP2 (Abcam ab3373). At the chosen time points inthe experimental design, the static cultures and cultures subjected tocontrolled hemodynamics were washed gently with 1×PBS, following whichthey were fixed with 4% paraformaldehyde for 30 minutes. The sampleswere stored in PBS at 4° C. until they were to be immunostained. Forimmunostaining, the samples were first permeabilized with 0.1% TRITON X(a nonionic surfactant) for 20 minutes and then washed with PBS andblocked with 5% goat serum. The incubation with primary antibodies wasat a dilution of 1:100 for 1 hour. After 3 washes with PBS with 1% BSA,the secondary antibody was added at a dilution of 1:500 for anotherhour. The samples were then washed with PBS plus 1% BSA and then mountedfor confocal imaging.

For imaging of the biliary activity at canalicular junctions, sectionsof the porous membrane of the TRANSWELL were washed with PBS andincubated with media containing 10 μM carboxy-2,7-dichlorofluoresceindiacetate (CDFDA) for 10 minutes. Samples were then washed with PBS andplaced on glass slide for confocal imaging.

Transmission Electron Microscopy:

Transmission electron microscopy was performed as described below inExample 13.

Cytochrome Activity Assays:

Hepatocytes were cultured in the cone-and-plate devices under static orcontrolled hemodynamic conditions for five days, and then treated with0.1% dimethyl sulfoxide (DMSO) or known inducers of cytochrome enzymes(3-methylcholanthrene and dexamethasone) for 48 hours. Porous membranesegments roughly 2 cm² in area were excised and transferred to standard24-well plates alongside corresponding static cultures. The cells wereincubated with 500 μl of hepatocyte media containing substrates fromcommercially available P450-GLO kits (kits for luminescent cytochromep450 assays) at the manufacturer-recommended concentrations. After 4hours, the media was transferred to 96-well plates and assayed forluminescent metabolites to reflect cytochrome p450 activity as per themanufacturer protocol. The ATP content of the cells in the same porousmembrane segments or static wells was then estimated by theCELLTITER-GLO assay (a kit for a luminescent cell viability assay) usingthe manufacturer's protocol, and the cytochrome values were normalizedto ATP content.

To assess CYP activity and induction responses of human hepatocytes, thecells were plated and cultured in the cone-and-plate devices andsubjected to controlled hemodynamics under the operating conditionsdescribed above or were cultured under static conditions (controls) for7 days before being exposed to either 0.1% DMSO or known CYP inducerdrugs phenobarbital (500 μM for static and 50 μM for devices) orrifampicin (25 μM for static and 2.5 μM for devices) for 72 hours. Thehepatocytes were then incubated with medium containing a cocktail of CYPsubstrates [(ethoxy resorufin (10 μM), midazolam (3 μM), bufuralolhydrochloride (10 μM), (S)-mephenytoin (50 μM), bupropion hydrochloride(100 μM), and diclofenac sodium (10 μM)] for 4 hours. The culturesupernatants were then collected and analyzed by HPLC for formation ofmetabolites to assess specific activity of specific CYP enzymes. Allvalues were normalized to protein content of the cells.

Gluconeogenesis Assays:

Primary rat hepatocytes isolated and plated as described above werecultured in the cone-and-plate devices under controlled hemodynamics for7 days. Hepatocytes were washed with PBS and incubated in glucose freemedia, with addition of substrates glycerol (2 mM) or lactate (20 mM)and pyruvate (2 mM) in the presence or absence of the regulatoryhormones insulin (2 nM) or glucagon (100 nM). After 4 hours, thesupernatants were collected and assayed for glucose content using thecolorimetric AMPLEX RED kit (a glucose/glucose oxidase assay kit, LifeTechnologies) as per manufacturer's instructions. The glucose valueswere normalized to the protein content of the cellular lysates.

MTT Assay:

To assess toxicity responses of human hepatocytes, the cells were platedand cultured in the cone-and-plate devices under hemodynamic conditionsusing the operating conditions described above or were cultured understatic conditions (controls) for 7 days before being exposed to either0.1% DMSO or known toxic drug chlorpromazine (0.1 μM, 1 μM and 10 μM)for 72 hours. Hepatocytes were then incubated with medium containing 1mg/ml of MTT reagent (thiazolyl blue tetrazolium bromide) for 1 hour,following which the cells were lysed in DMSO to release the formazanblue dye formed. The solution was transferred to a 96 well plate and theabsorbance was read at 595 nm.

Live-Dead Staining:

To assess toxicity responses of human hepatocytes, the cells were platedand cultured in the cone-and-plate devices under hemodynamic conditionsunder the operating conditions described above for 7 days or werecultured under static conditions (controls) before being exposed toeither 0.1% DMSO or known toxic drug chlorpromazine (0.1 μM, 1 μM and 10μM) for 72 hours. At the end of the treatment period, the hepatocyteswere washed with PBS and then incubated in LIVE/DEADviability/cytotoxicity reagent (Invitrogen) at a concentration of 2 μMcalcein AM and 4 μM ethidium homodimer-1 (EthD-1) for 30 minutes. Cellswere then mounted between glass coverslips and imaged using a confocalmicroscope.

miRNA122 Assay:

Rat hepatocytes were plated and cultured in the cone-and-plate devicesunder controlled hemodynamic or were cultured under static conditions(controls) using the operating conditions described above for 7 days.The hepatocytes were then washed with PBS and incubated with serum freehepatocyte medium with or without known toxic drug chlorpromazine (CPZ)at two different concentrations (1 μM and 10 μM) for 4 hours.Supernatants from the cells were collected and microRNA extraction wasperformed using the MIRNEASY serum/plasma kit (a kit for extractingmicroRNA, Qiagen). The cDNA was prepared by using the MISCRIPTII RT kit(a kit for preparing cDNA, Qiagen) and samples quantified using theMISCRIPT SYBR GREEN PCR kit (a kit for quantifying cDNA, Qiagen),following the manufacturer's instructions.

Results

(ii) Controlled Hemodynamics Maintain Hepatocyte Phenotype, PolarizedMorphology and Transporter Localization Relative to Traditional StaticMonoculture Conditions.

Freshly isolated rat primary hepatocytes were obtained and plated incollagen gel sandwiches on porous membranes. After 1 day, cultures wereeither continued under standard static conditions in a CO₂ incubator at37° C. or introduced into the hemodynamic flow technology and maintainedunder controlled hemodynamics at pre-determined indirect shear rates of0.6 dynes/cm². Media was changed every 48 hours in static cultures andthe devices were continuously perfused. After 7 days, the cultures wereremoved and fixed with 4% paraformaldehyde before immunostaining withantibodies for the hepatocyte differentiation markers E-cadherin andHNF-4α, and visualized by confocal microscopy. E-cadherin stainingpatterns in static collagen gel sandwich cultures (FIG. 20A) displayedhigher levels of cytoplasmic E-cadherin confirmed and quantified bymorphometric analysis (adjacent graphs) and disrupted peripheralmembrane distribution. Under controlled hemodynamics (FIG. 20B),hepatocytes exhibited a more differentiated morphology characterized bydistinct peripheral membrane localization and lower cytoplasmic levelsof E-cadherin. The staining pattern of the HNF4α showed a distinctdifference in localization patterns with the cells in static cultureshaving a more diffuse staining pattern by 7 days (FIG. 20C) while thecells under controlled hemodynamics retained staining confined to thenucleus (FIG. 20D), similar to what is seen in vivo. Polarizedmorphology and canalicular localization of the transporter multi drugresistant protein-2 (MRP-2) that appears after 5-7 days of culture incollagen gel sandwiches is lost in static cultures by day 14 (FIG. 20E)but the canalicular network patterns are stable and extensive undercontrolled hemodynamics (FIG. 20F). Day 14 cultures maintained undercontrolled hemodynamics co-stained for MRP-2 and HNF-4α (FIG. 21A)alongside sections from rat in vivo liver (FIG. 21B) show very similarstaining patterns. Transmission electron microscopy images of day 7cultures under controlled hemodynamics (FIG. 21C) demonstrate theretention of subcellular components such as rough and smooth endoplasmicreticulum and mitochondria in addition to confirming the presence ofbile canaliculi and tight junctions.

(ii) Controlled Hemodynamics Results in Retention of Hepatocyte-SpecificFunction in Rat Hepatocytes in a Collagen Gel Configuration Relative toStatic Cultures Over 14 Days.

Hepatocytes were cultured under static or controlled hemodynamics (0.6dynes/cm²) for 2 weeks and media sampled at 4, 7, 11, and 14 days.Assays for urea and albumin were performed on the media and the valueswere normalized to production rates over 24 hours per million cellsbased on the initial number of plated cells. Hepatocyte functionreflected by secreted albumin estimated from media samples at varioustime points over 14 days and expressed as μg/10⁶ plated hepatocytes/day(FIG. 22A), showed significantly higher levels (3-4 fold) undercontrolled hemodynamics (solid line) as compared to static cultures(dashed line) (Day 7: 97.96±11.34 vs. 25.84±8.22, p=0.00001; Day 14:87.80±8.62 vs. 33.93±4.39, p=0.0001). Urea secretion (FIG. 22B) byhepatocytes expressed as μg/10⁶ plated hepatocytes/day under controlledhemodynamics (solid line) was also found to be at 4-5 fold higher levelsthan static cultures (dashed line) consistently over two weeks inculture (Day 7: 622.78±33.96 vs. 139.76±13.37, p=2.7×10⁻⁹; Day 14:667.71±84.37 vs. 178.68±6.13, p=1×10-6).

(iii) Controlled Hemodynamics Differentially Regulates the Expression ofPhase I and Phase II Metabolic Genes and Proteins Compared to StaticCultures.

Hepatocytes were cultured under static or controlled hemodynamics (0.6dynes/cm²) for 7 days. QRT-PCR was performed for select metabolic genes(Table 3) on RNA samples at day 7 from these conditions. All values werenormalized to day 7 static cultures. Hepatocytes cultured undercontrolled hemodynamics resulted in gene expression levels that wereconsistently higher than in static cultures (n=11, Fold changes relativeto static cultures: Cyp1A1˜54, p=0.0003; Cyp1A2˜64, p=0.005, Cyp2B1˜15,p=0.001: FIG. 23A, Cyp2B2˜2.7, p=0.09 and Cyp3A2˜4, p=0.075: FIG. 23B)and closer to in vivo levels. Interestingly, the expression levels ofthe gene for the Pi subunit of phase II enzyme GST, known to increase instatic cultures over time, was lower in both in vivo liver (˜4.9 fold,p=0.152) and hepatocytes cultured under controlled hemodynamics (−2.3fold, p=0.025) compared to static cultures (FIG. 23C).

Hepatocytes were cultured under static or controlled hemodynamics (0.6dynes/cm²). Cell cultures were taken down at 4, 7, 11 and 14 days andcell lysates were obtained as described in the methods section,normalized to total protein, and equivalent samples were loaded and runon SDS page gels before probing with antibodies for the phase II enzymeUGT1 A1 and β-actin (for normalization). Western blots (FIG. 23D)demonstrate that UGT1 A1 is upregulated under controlled hemodynamics ascompared to static conditions at all the time points over 2 weeks inculture. In the same experiment, part of the porous membrane of theTRANSWELL from 14 day cultures under controlled hemodynamics was fixedwith 4% paraformaldehyde and stained for HNF-4a and the canaliculartransporter protein MRP-2, demonstrating retention and localization ofMRP-2 along the canalicular junctions between the hepatocytes (FIG.21A). The remainder of the membrane was excised after removal from thedevice and immediately incubated with the substratecarboxy-2,7-dichlorofluorescein diacetate (CDFDA). The cells were imagedby confocal microscopy over a time window of 20 minutes to observe thebreakdown of the substrate into carboxy-2,7-dichlorofluorescein (CDF)and its active secretion into the bile canalicular structures (seen inFIG. 21C). The pattern was very similar to that of sectioned samples ofin vivo liver immunostained with antibodies to MRP-2 and HNF-4a (FIG.21B).

(iv) Rat Hepatocytes Cultured Under Controlled Hemodynamics Display aHigher Level of Basal and Inducible Cytochrome p450 Activity than StaticCultures at More In Vivo-Like Concentrations.

To validate that the increase in metabolic genes and proteins translatedto changes in metabolic activity, primary rat hepatocytes were culturedas described earlier in the cone-and-plate devices under controlledhemodynamics (0.6 dynes/cm²) and in static collagen gel cultures. After5 days, they were either left untreated or treated with 0.1% DMSO, 1A/1Binducer 3-Methyl Cholanthrene (3-MC, 1 μM in static and 0.1 μM undercontrolled hemodynamics) or 3A inducer dexamethasone (50 μM in staticand 02.5 μM under controlled hemodynamics). After 48 hours, on day 7,segments of the porous membrane from the devices containing hepatocytescultured under controlled hemodynamics that were roughly 2.0 cm² in areawere excised and transferred to standard 24-well plates and treated withsubstrates for the Cyp p450 enzymes in parallel to corresponding staticcultures treated with the different agents. Cytochrome p450 assays weredone on day 7 using commercially available P450-GLO kits. After 4 hoursthe media was transferred to 96-well plates and assayed for luminescentmetabolites to reflect cytochrome p450 activity. Values were normalizedto the ATP content of the cells assessed by CELLTITER-GLO assay in orderto get an accurate representation of live cells and avoid anyconfounding effects of the collagen gels on total protein measurements.

Basal activity level of the cytochrome p450 enzymes (FIG. 24A) inuntreated cultures was upregulated by controlled hemodynamics comparedto static (1A˜15 fold, 1B˜9 fold and 3A˜5 fold). In spite of higherlevels of basal activity, under controlled hemodynamics the response toclassical inducers (FIG. 24B) was well maintained (1A/1B response toDMSO vs. 3—MC-4.87 vs. 133.06; 3A response to DMSO vs.Dexamethasone—11.64 vs. 57.53).

While initially measuring the Cyp activity to confirm the enhanced geneexpression that was noted under controlled flow, 50 μM dexamethasone,the concentration recommended for inducing static cultures, was toxic inthis system. As a result the concentration of the dexamethasone wasdecreased to 1 μg/ml in order to get an inductive response, a level thatcorrelates well with plasma concentrations seen in vivo in rats.Similarly, induction responses for 3-MC were also seen at 10-fold lowerlevels under controlled hemodynamics.

To confirm the presence of transporter activity under controlledhemodynamics, TRANSWELL filter segments from the devices were incubatedwith the substrate carboxy-2,7-dichlorofluorescein diacetate (CDFDA).The compound was broken down to the fluorescent form CDFCarboxy-2,7-Dichlorofluorescein which was actively secreted out into thecanalicular spaces demonstrating active canalicular transport (FIG.24C).

The data described above are the result of experiments carried out toevaluate the effect of exposing hepatocytes to controlled hemodynamicsin order to restore their phenotype more similar to that observed invivo. These experiments used standard media formulations routinely usedin static culture in order to allow for side by side comparison with thestatic collagen gel cultures and identify the selective benefits ofcontrolled hemodynamics. In the course of these experiments, hepatocytescultured under these controlled hemodynamic conditions demonstratedenhanced in vivo-like phenotype and function and were more responsive toinducers such as dexamethasone and 3-MC. However, some accumulation oflipids was also observed in hepatocytes cultured with the concentrationsof glucose (17.5 mM) and insulin (2 μMol) which are used routinely forassays in static systems. It was discovered that when hepatocytes arecultured under controlled hemodynamic conditions as described herein,much lower concentrations of glucose and insulin, similar to theconcentrations observed in healthy individuals in vivo, can be used. Thedata indicate that these lower concentrations of glucose (5.5 mM) andinsulin (2 nM) further enhance hepatocyte function and metabolicactivity. Moreover, hepatocytes can be cultured under controlledhemodynamics in media containing the higher concentrations of glucoseand insulin in order to create a model of fatty liver disease, asexplained further in the following Example.

(v) Primary Rat Hepatocytes Cultured Under Controlled HemodynamicsDemonstrate Responsiveness to Insulin and Glucagon.

Primary rat hepatocytes isolated and plated as described above werecultured in the cone-and-plate devices under controlled hemodynamics for7 days prior to washing with PBS and incubation with the substratesglycerol (2 mM) or lactate (20 mM) and pyruvate (2 mM) either in thepresence or absence of the regulatory hormones insulin (2 nM) orglucagon (100 nM). Glucose levels measured in the supernatant after 4hours by the AMPLEX RED assay showed that in the absence of a substrate,insulin decreased glucose levels by 27% while glucagon increased it by51%. In the presence of the substrate glycerol, glucose produced by thehepatocytes increased by 67%. Addition of glucagon increased glucoselevels by further 15% while insulin decreased glucose levels by 38%.When lactate and pyruvate were used as substrates, glucose produced bythe hepatocytes increased in the presence of glucagon by 80% whileinsulin decreased glucose levels by 25%. These data are summarized inTable 6.

TABLE 6 Effect of Insulin Effect of Glucagon Substrate (% Change) (%Change) No substrate −27% +51% Glycerol (+67%) −38% +15%Lactate/Pyruvate −25% +80%(vi) Cryopreserved Human Hepatocytes Cultured Under ControlledHemodynamics Demonstrate Induction Responses to Phenobarbital andRifampicin at In Vivo Level Concentrations.

Human hepatocytes were cultured in the cone-and-plate devices undercontrolled hemodynamics under the operating conditions described aboveor were cultured under static conditions (controls) for 7 days beforebeing exposed to the known CYP inducer drugs phenobarbital (500 μM infor static conditions and 50 μM for controlled hemodynamic conditions)or rifampicin (25 μM in for static conditions and 2.5 μM for controlledhemodynamic conditions) for 72 hours. The hepatocytes were then washedwith PBS and incubated with medium containing a cocktail of CYPsubstrates as described above for 4 hours. The culture supernatants werethen collected and analyzed for formation of metabolites to assessspecific activity of specific CYP enzymes. Results were normalized toprotein content of the cells and expressed as pmol/min/mg of protein.Vehicle treated controls with DMSO 0.1% exhibited higher levels ofCYP2B6, CYP2C9 and CYP3A4 in under controlled hemodynamic conditions ascompared to static conditions (7.7 vs. 4.6, 4.6 vs. 0.5 and 7.6 vs. 0.7pmol/min/mg of protein, respectively). Treatment with phenobarbital atthe lower concentration (50 uM) under controlled hemodynamic conditionscompared to higher concentration under static conditions (500 μM) alsoresulted in comparable or higher levels of enzyme activities of CYP2B6,CYP2C9 and CYP3A4 (45.9 vs. 34.3, 16.3 vs. 0.9 and 16.3 vs. 3.8pmol/min/mg of protein, respectively). Similarly, treatment withrifampicin at the lower concentration (2.5 μM) under controlledhemodynamic conditions compared to the higher concentration in staticconditions (25 μM) also resulted in comparable or higher levels ofenzyme activities of CYP2B6, CYP2C9 and CYP3A4 (87.3 vs. 131.1, 1.4 vs.16.0 and 11.5 vs. 23.1 pmol/min/mg of protein, respectively). Theseresults are depicted in FIG. 35.

(vii) Cryopreserved Human Hepatocytes Cultured Under ControlledHemodynamics Demonstrate Toxicity Responses to Chlorpromazine at In VivoLevel Concentrations.

Cryopreserved primary human hepatocytes thawed and plated as describedabove were cultured in the cone-and-plate devices under controlledhemodynamics or were cultured under static conditions (controls) for 7days before being exposed to different concentrations of chlorpromazine(0.1 μM, 1 μM, and 10 μM) or vehicle control for 72 hours. Live-deadstaining was performed on the hepatocytes with ethidium-calcein stain.Hepatocytes were also incubated with MTT reagent for 1 hour to assessviability. RNA was extracted from additional segments and RT-PCR wasperformed to assess selected toxicity and metabolic genes. Hepatocytescultured under static conditions did not exhibit any toxicity at all theconcentrations tested. However hepatocytes cultured under controlledhemodynamics demonstrated dose-dependent toxicity with 30.3% toxicity at1 μM and 46.4% toxicity at 10 μuM (FIG. 36B). At 1 μM, the toxicity tothe hepatocytes cultured under controlled hemodynamics devices was alsodetected by live-dead staining (FIG. 36A).

RT-PCR demonstrated upregulation of various oxidative stress relatedtoxicity genes at 1 μM chlorpromazine under controlled hemodynamicconditions relative to static controls (8.3-fold for glutathionereductase (GSR), 5.5-fold for thioredoxin reductase 1 (TXNRD1), 6.9-foldfor sorbitol dehydrogenase (SORD), and 2.8-fold for APEX nuclease(multifunctional DNA repair enzyme)). Concomitantly, certain metabolicgenes were also upregulated under controlled hemodynamic conditionsrelative to static controls (17.8-fold for cytochrome p450 family 1member A2 (CYP1A2), 8.4-fold for cytochrome p450 family 1 member A1(CYP1A1), and 5.6-fold for Cytochrome p450 family 2 member B6 (CYP2B6).These results are depicted in FIG. 37. The results shown in FIG. 37 usedprimary human hepatocytes from KalyCell Donor #B0403VT.

These data show that primary human hepatocytes display toxic responsesto chlorpromazine at clinical plasma C_(max) concentrations undercontrolled hemodynamic conditions. These toxic responses are associatedwith the upregulation of oxidative stress-related genes and certainmetabolic genes.

(viii) Primary Rat Hepatocytes Cultured Under Controlled HemodynamicsDemonstrate Acute Toxicity and Release miRNA122 in Response toChlorpromazine Exposure at In Vivo Level Concentrations.

Primary rat hepatocytes isolated and plated as described above werecultured in the cone-and-plate devices under controlled hemodynamicconditions or were cultured under static conditions (controls) for 7days. The hepatocytes were washed with PBS and immediately incubatedwith either vehicle (distilled water) or chlorpromazine (1 μM) for 4hours. The supernatant was collected and miRNA 122 levels were measuredas described above. It was seen that under static conditions,chlorpromazine at 1 μM did not cause any change in miRNA 122 levels inthe supernatants compared to vehicle controls. By contrast, hepatocytescultured under controlled hemodynamic conditions and incubated withchlorpromazine (1 μM) for 4 hours released miRNA at significantly higherlevels (6-fold over vehicle controls). These results are depicted inFIG. 38.

(ix) Primary Rat Hepatocytes Cultured Under Controlled HemodynamicsDemonstrate Sublethal Toxicity and Exhibit Cholestatic Changes inResponse to Troglitazone Exposure at In Vivo Level Concentrations.

Primary rat hepatocytes isolated and plated as described above werecultured in the cone-and-plate devices under controlled hyemodynamicconditions for 5 days before being exposed to 4 μM or 40 uM troglitazonefor 48 hours. The hepatocytes were washed with PBS and immediatelyincubated with the substrate 10 uM carboxy-2,7-dichlorofluoresceindiacetate (CDFDA). The cells were imaged by confocal microscopy during a20-min exposure to the nonfluorescent substrate CDFDA to allow for thehydrolysis of the substrate to the highly fluorescent Mrp-2 substratecarboxy-2,7-dichlorofluorescein (CDF) and its active secretion into thebile canalicular structures. At 4 uM, troglitazone was found to causechanges in the canalicular pattern with visibly dilated canalicularstructures. These changes were much more prominent and extensive at 40uM troglitazone (FIG. 39). The toxic response of rat hepatocytes totroglitazone at in vivo/clinical plasma C_(max) concentrations whencultured under controlled hemodynamic conditions was associated withupregulation of oxidative stress-related genes and compensatoryupregulation of MRP3 and MRP4 genes (FIG. 40).

(x) Primary Dog Hepatocytes Cultured Under Controlled HemodynamicsDemonstrate Retention of Polarized Morphology and Exhibit HigherExpression of Key Metabolic Genes Relative to Static Cultures.

Freshly isolated canine hepatocytes were cultured in the cone-and-platedevices under controlled hemodynamic conditions under operatingconditions similar to those described above for human hepatocytes orwere cultured under static conditions (controls). After 7 days, cultureswere fixed and stained with phalloidin and Draq5 for actin cytoskeletonand nucleus, respectively. RNA was collected from cells and RT-PCR wasperformed for specific metabolic genes. Canine hepatocytes were seen toretain polarized morphology with polygonal shape at 7 days and toexpress CYP1A1 and CYP3A12 at significantly higher levels than staticcontrols (6.7- and 7.4-fold respectively). These results are depicted inFIG. 41.

Example 13 An In Vitro Model for Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is the most common cause ofliver dysfunction and is associated with obesity, insulin resistance,and type 2 diabetes. The changes in the fatty liver progress from earlyaccumulation of fat vesicles within hepatocytes (hepatic steatosis) tosubsequent loss of liver metabolic function and inflammatory changes,ultimately leading to fibrosis and cirrhosis. Animal in vivo models offatty liver disease have successfully used either high fat diets or lowfat, high carbohydrate diets that induce the hyperglycemia andhyperinsulinemia reflective of the diabetic milieu to inducetriglyceride buildup. However in vitro models typically use onlyoverloading with free fatty acids (oleic, palmitic or linoleic acid) toinduce fatty changes and may not capture the de novo hepatocyte responseto the high levels of glucose and insulin that may play a critical rolein the pathogenesis of the disease. Static hepatocyte cultures are alsoknown to have a markedly decreased insulin response and standard culturemedias typically require high non-physiological levels of the hormonefor basic hepatocyte survival and function. The model described herein,by contrast, preserves a more physiological hepatocyte response to drugsand hormones and allows us to maintain basic liver function at closer toin vivo concentration levels of glucose and insulin (as described abovein Example 12), and furthermore allows us to elicit the pathologicresponse seen in fatty liver by creating a diabetic-like milieucharacterized by high glucose and insulin levels.

Methods:

(i) Animal Surgery and Hepatocyte Isolation

Animal surgery and hepatocyte isolation were performed as describedabove in Example 12.

(ii) Cell Culture and Device Operating Conditions

Healthy Hepatocyte Culture Media:

The healthy hepatocyte culture media contained base media of DMEM/F12containing low glucose (5.5 mM), supplemented by fetal bovine serum (10%at the time of plating and reduced to 2% for maintenance after 24hours). Additionally, the media contained gentamycin (50 mg/ml), ITS(insulin, transferrin, and selenium; insulin concentration of 2 nM), 1%non-essential amino acids (NEAA), 1% GLUTAMAX (a media supplementcontaining L-alanyl-L-glutamine), and dexamethasone (1 μM at plating and250 nM for maintenance after 24 hours for the data shown in FIGS. 25 and26; 100 nM throughout the experiment for the data shown in FIGS. 27-34).

Media to Induce Fatty Liver Changes (“Fatty Liver Media”):

The culture media used to induce fatty liver changes contained basemedia of DMEM/F12 containing high glucose (17.5 mM), supplemented byfetal bovine serum (10% at the time of plating and reduced to 2% formaintenance after 24 hours). The media also contained gentamycin (50μg/ml), ITS (insulin concentration 2 μMol), 1% NEAA, 1% GLUTAMAX, anddexamethasone (1 μM at plating and 250 nM for maintenance after 24 hoursfor the data shown in FIGS. 25 and 26; 100 nM throughout the experimentfor the data shown in FIGS. 27-34).

Collagen Coating and Plating:

Collagen solution was made as described above in Example 12. The lowersurfaces of the porous membranes of 75 mm TRANSWELLS (polycarbonate, 10μm thickness and 0.4 μm pore diameter, no. 3419, Corning) were coatedwith 300 μl of the collagen solution. After allowing an hour for thesolution to gel, the surfaces were washed with DPBS, hepatocytes wereplated at a seeding density of 125,000 viable cells/cm², and a secondlayer of collagen gel added after 4 hours. After 1 hour, the TRANSWELLSwere inverted and placed into cell culture dishes, and media was added(9 ml in the lower volume and 6 ml in the upper volume). After 24 hours(i.e., on day 2 of the experiments), the media was changed tomaintenance media (the healthy or fatty liver media described above) andthe Petri dishes were placed in the cone-and-plate hemodynamic flowdevice, and controlled hemodynamics were applied to the surface of theporous membrane of the TRANSWELL in the upper volume. In someexperiments, the maintenance media contained 1.5 μM pioglitazone in 0.1%DMSO vehicle or the 0.1% DMSO vehicle alone. The cells were culturedunder controlled hemodynamics until day 7, when hepatocytes wereexamined using the assays described below.

Operating Conditions:

The shear stress was calculated as described above in Example 12. Arange of applied shear stress conditions, generated by altering mediaviscosity and cone speed, and resulting in rates within an order ofmagnitude of the value predicted from literature (0.1 to 6 dynes/cm²)were used. These were correlated with different transport profiles ofreference dye horse radish peroxidase dye across the membrane. Cultureswere run for 7 days and assessed for fatty liver changes.

(iii) Measurement of Fatty Liver Changes:

To examine changes occurring in the fatty liver model against healthycontrols the following were evaluated:

-   -   (a) Changes in metabolic and insulin/glucose/lipid pathway genes        (RT-PCR);    -   (b) Accumulation of intracellular lipids within hepatocytes by        Oil Red O assay, Nile red staining, and measurement of total        triglycerides;    -   (c) Changes in differentiated function of hepatocytes (urea and        albumin secretion);    -   (d) Changes in metabolic activity (Cytochrome p450 assays); and    -   (e) Morphological changes within hepatocytes by transmission        electron microscopy (TEM).

RT-PCR and urea and albumin assays were performed as described above inExample 12.

Staining Methods:

Hepatocyte TRANSWELL membrane sections were permeabilized in 0.1%Triton-X diluted in PBS for 20 minutes and washed thrice in PBS for fiveminutes each. Samples were then blocked in 5% goat serum, 0.2% blottinggrade non-fat dry milk blocker, and 1% BSA in PBS for 45 minutes. Thesamples were then washed thrice in 0.1% BSA in PBS and incubated with1:5000 dilution of Nile red (1 mM stock), 1:1000 DRAQ5 (a fluorescentDNA dye; Cell Signalling), 1:500 ALEXA FLUOR 488 conjugated phalloidin(Life Technologies), and 1% BSA in PBS for thirty minutes and protectedfrom light. The samples were washed in 0.1% BSA in PBS thrice for fiveminutes each and mounted on glass cover slips using PROLONG GOLDantifade mounting media (an antifade reagent; Invitrogen). The sampleswere imaged on a Nikon C1+ Confocal System microscope.

Transmission Imaging Microscopy (TEM):

Segments of the porous membranes from TRANSWELLS containing hepatocytescultured under healthy or steatotic conditions for 7 days were washedwith PBS before fixing in a solution containing 4% paraformaldehyde and2% glutaraldehyde for 1 hour. The samples were then sent to be processedfor TEM at the University of Virginia imaging center. TEM images wereevaluated for accumulation of lipid within the hepatocytes, theappearance of subcellular organelles such as mitochondria and smooth andrough endoplasmic reticulum, retention of polarized morphology, and bilecanaliculi.

Oil Red O Assay:

Accumulation of intracellular lipids within hepatocytes was assessed byadapting and modifying a commercially available Steatosis ColorimetricAssay Kit (Cayman Chemical). At the end of the culture period, 2 cm²sized porous membrane segments containing the hepatocytes from devicesunder healthy and steatotic conditions were washed with PBS and fixed in4% paraformaldehyde for 30 minutes. These porous membrane segments werethen washed with PBS, dried completely and incubated with 300 μl of OilRed O working solution for 20 minutes in 24 well plates. The porousmembrane segments were then washed repeatedly with distilled water 7-8times followed by two five minute washes with the wash solution providedin the Steatosis Colorometric Assay Kit. Dye extraction solution (300μl) was added to each well and the plates were incubated on an orbitalshaker for 15-30 minutes under constant agitation. The solution was thentransferred to clear 96-well plates and absorbance was read at 490-520nm in a spectrophotometer.

Measurement of Total Triglycerides:

Triglyceride content was assessed using a commercially availablecolorimetric assay kit (Cayman Triglyceride Colorimetric Assay Kit, Cat#10010303). At the end of the treatment period, cells were collectedfrom the porous membranes by scraping with a rubber policeman and PBS,after which they were centrifuged (2,000×g for 10 minutes at 4° C.). Thecell pellets were resuspended in 100 μl of cold diluted Standard Diluentfrom the triglyceride assay kit and sonicated 20 times at one secondbursts. The cell suspension was then centrifuged at 10,000×g for 10minutes at 4° C. The supernatant was removed and used for the assay asper the manufacturer's protocol and normalized to protein content fromthe same samples.

Cytochrome Activity Assays:

Hepatocytes were cultured in the cone-and-plate devices under healthyand steatotic conditions for 7 days. Porous membrane segments roughly 2cm² in area were excised and transferred to standard 24-well platesalongside corresponding static cultures. The cells were incubated with500 μl of healthy hepatocyte media containing substrates fromcommercially available P450-GLO kits at the manufacturer-recommendedconcentrations. After 4 hours, the media was transferred to 96-wellplates and assayed for luminescent metabolites to reflect cytochromep450 activity as per the manufacturer protocol. The ATP content of thecells in the same porous membrane segments or static wells was thenestimated by the CELLTITER-GLO assay using the manufacturer's protocol,and the cytochrome values were normalized to ATP content.

Results:

Nile Red Staining:

FIGS. 27A and B show staining of hepatocytes cultured in the healthy(FIG. 27A) or fatty liver (FIG. 27B) media with Nile red, phalloidn, andDRAQ5. As can be seen in FIG. 27B, the hepatocytes cultured in the fattyliver media (containing high concentrations of glucose and insulin)accumulate a large number of lipid droplets.

Transmission Electron Microscopy:

Hepatocytes cultured in the fatty liver media were also examined bytransmission electron microscopy. As shown in FIG. 28, hepatocytescultured under these conditions accumulate lipid. A large lipid dropletis indicated in the hepatocyte on the left side of the image. Gapjunctions between two hepatocytes are also shown, demonstrating thepolarized morphology.

Total Lipid and Total Triglycerides:

As shown in FIG. 29, total lipid (FIG. 29A) and total triglycerides(FIG. 29B) were both significantly increased in hepatocytes culturedunder the high glucose/high insulin fatty liver conditions in thepresence of liver-derived hemodynamics. Oil red O quantificationindicated that the total lipid was raised in the disease cultures byabout 3-fold as compared to the healthy cultures.

Gene Expression:

Glycerol 3-phosphate acyltransferase (GPAT) is a key enzyme involved intriglyceride synthesis and known to upregulated and contribute tosteatosis and fatty liver. As shown in FIG. 25, primary rat hepatocytescultured under controlled hemodynamics in the devices when exposed topathological conditions (n=9) of high insulin (2 μMol) and high glucose(17.5 mMol) exhibit a significantly higher expression the GPAT gene(p=0.04) compared to those cultured under healthy physiological levels(n=6) of insulin (2 nMol) and glucose (5.5 mMol) in the media. Theresults are expressed as fold increase over standard static cultures incollagen gel sandwiches (2 μMol insulin and 17.5 mMol glucose).

Similar results are shown in FIG. 30B for hepatocytes cultured undercontrolled hemodynamics in healthy or fatty liver media containing alower concentration of dexamethasone. The hepatocytes cultured in thehigh insulin/high glucose (fatty liver) media exhibited significantlyhigher levels of GPAT expression as compared to hepatocytes cultured inthe healthy media containing lower levels of insulin and glucose. Asshown in FIG. 30A, hepatocytes cultured under controlled hemodynamics inthe high insulin/high glucose media also exhibited significantly higherlevels of expression of sterol regulatory element-binding protein(SREBP), another key gene responsible for lipogenisis, as compared tohepatocytes cultured in the healthy media.

These steatotic changes were accompanied by concomitant metabolicchanges. Of all the key metabolic enzymes, the cytochrome p450 3A familyis responsible for the metabolism of a majority of drugs. As shown inFIG. 26, primary rat hepatocytes cultured under controlled hemodynamicsin the devices with healthy physiological levels (n=6) of insulin (2nMol) and glucose (5.5 mMol) in the media, exhibit a significantlyhigher expression level of the key metabolic enzyme cytochrome p450 3a2(Cyp3A2; p=0.03), compared to those cultured under pathologicalconditions (n=9) with high insulin (2 μMol) and high glucose (17.5 mMol)levels. Both the healthy and pathological fatty liver levels undercontrolled flow are many fold higher than static cultures in collagengel sandwiches (2 μM insulin and 17.5 mMol glucose).

Similarly, as shown in FIG. 31A, expression of a number of phase Ienzymes involved in drug metabolism are differentially regulated underlow and high glucose/insulin conditions. Under hemodynamic flow,hepatocytes under healthy media conditions maintained high levels ofmRNA expression of Cyp1a1, Cyp 2b1, 2b2, Cyp3a2, and (20, 90, 30 and40-fold higher than traditional static cultures respectively), whereasCyp 2b2 and Cyp 3a2 levels in hepatocytes cultured in the fatty livermedia were decreased by 9 and 12 fold compared to healthy.

Cyp Activity:

As shown in FIG. 31B, the activities of CYP3A2 and CYP1A1 were alsoreduced 3-6-fold under the high insulin/glucose fatty liver conditionscompared to healthy, as measured by the p45glo assay.

Pioglitazone Treatment:

Pioglitazone, a drug used to treat steatosis, was tested in the fattyliver model to determine if it could reverse the lipid accumulation andmetabolic changes induced by the high insulin/glucose fatty liver media.The pioglitazone was added to the media at a concentration of 1.5 μM, aconcentration selected based on the therapeutic C_(max) observed forpioglitazone in vivo. Pioglitazone was effective in reducing the lipidbuildup and triglyceride content while restoring metabolic geneexpression under the disease conditions. As shown in FIG. 32, Nile redstaining indicates that treatment with pioglitazone at in vivotherapeutic concentrations decreases lipid droplet formation understeatotic conditions. Pioglitazone also reduced total triglyceridecontent of hepatocytes cultured in the high insulin/glucose media tolevels similar to those seem in the hepatocytes cultured under healthyconditions (FIG. 33). Moreover, as shown in FIG. 34, pioglitazonerestored the expression of metabolic genes such as Cyp3A2 which aredepressed by the high insulin/glucose disease conditions.

CONCLUSIONS

In summary, a system was developed that preserves in vivo-likehepatocyte phenotype and response, to create a model of hepaticsteatosis by inducing pathological steatotic changes in the presence ofa high glucose/insulin milieu. Rat hepatocytes under controlledhemodynamics retain their response to insulin and glucose, andhepatocytes cultured under hemodynamic flow develop steatotic changeswhen cultured in high glucose and insulin (‘disease’) conditions. Thesteatosis is mediated via de novo lipogenesis with upregulation of twokey genes (SREBP and GPAT), and the increase in lipid accumulation andtriglyceride content is accompanied by a concomitant decrease inmetabolic gene expression and activity. Treatment with the PPAR-γagonist pioglitazone helps prevent the buildup of lipid and loss ofmetabolic activity under the high glucose and insulin conditions. Thesedata demonstrate a novel and important new in vitro model of dietinduced non-alcoholic fatty liver disease (NAFLD) for which nonecurrently exist.

Example 14 An Inducible Pluripotent Stem Cell (iPSC)-Derived HumanHepatocyte System

Hepatocytes derived from inducible pluripotent stem cells (iPSCs) offera potential solution for eliminating variability and studying genotypicvariation in drug response but have not found widespread acceptance onaccount of the fetal phenotype and inadequate metabolic profile theyexhibit in standard, static culture systems. The data described above inExample 12 demonstrate that primary rat and human hepatocytes, which areknown to rapidly dedifferentiate under static culture conditions, stablyretain a mature differentiated phenotype when cultured under controlledhemodynamic conditions, resulting in a more physiologic drug and hormoneresponse. It was discovered that iPSCs respond similarly whenphysiological properties such as flow, hemodynamics and transport aremaintained and exhibit the differentiated liver phenotype and responseto drugs that they exhibit in vivo.

Methods

(i) iPSC-Derived Hepatocytes

iPSC-derived Hepatocytes were purchased from Cellular DynamicsInternational.

(ii) iPSC-Derived Hepatocyte Culture Media

The iPSC-derived hepatocyte culture media for static cultures was as perthe vendors recommendations. For cells cultured under controlledhemodynamic conditions in the cone-and-plate devices, a base media ofWilliams E medium supplemented by fetal bovine serum (10%) anddexamethasone (1 μM) at the time of plating was used. Maintenance mediawas used after 24 hours that did not contain FBS but was supplementedwith bovine serum albumin (0.125%). The media also contained gentamycin(25 μg/ml), ITS (insulin concentration 2 nMol), 1% NEAA, 1% GLUTAMAX,HEPES (30 mM) and dexamethasone (100 nM).

(iii) Collagen Coating and Plating

The collagen coating and plating conditions were identical to thosedescribed above in Example 12 for primary human hepatocytes. TheiPSC-derived hepatocytes were dissociated and plated as per the vendor'sprotocols using the recommended media. iPSC-derived hepatocytes werecultured under static conditions or were transferred into thecone-and-plate devices after 24 hours for further culture undercontrolled hemodynamic conditions.

Results

(i) Hepatocytes Derived from Inducible Pluripotent Stem Cells (iPSCs)Cultured Under Controlled Hemodynamic Conditions Retain PolarizedMorphology and Exhibit Higher Expression of Key Metabolic Genes Relativeto Static Cultures.

iPSC-derived hepatocytes cultured in the cone-and-plate devices undercontrolled hemodynamics for 10 days retain polarized morphology (FIG.42) and exhibit higher expression of key metabolic genes relative tostatic cultures (104-fold for CYP1A1, 91-fold for CYP1A2, 8.8-fold forCYP3A4, 8.2-fold for CYP2B6, 2.3-fold for CYP2C9 and 2.3-fold forCYP2D6). Expression of the constitutive androstane receptor CAR was6.0-fold higher than cells cultured under static conditions and theliver-specific protein albumin was at 2.2-fold higher levels than incells cultured under static conditions. These results are depicted inFIG. 43.

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When introducing elements of the present invention or the preferredembodiments(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above methods without departingfrom the scope of the invention, it is intended that all mattercontained in the above description and shown in the accompanyingdrawings shall be interpreted as illustrative and not in a limitingsense.

What is claimed is:
 1. A method for mimicking a tumor microenvironmentin vitro, the method comprising: adding a culture medium to a cellculture container; plating at least one tumor cell type on a firstsurface of a porous membrane within the cell culture container; andindirectly applying a shear stress upon the at least one tumor cell typeby applying the shear stress upon a second surface of the porousmembrane, the shear stress resulting from flow of the culture mediuminduced by a flow device, the flow mimicking flow to which the tumorcells are indirectly exposed in vivo in the tumor microenvironment. 2.The method of claim 1, wherein the method further comprises plating atleast one stromal cell type on the first surface of the porous membrane.3. The method of claim 1, wherein the porous membrane is suspended inthe cell culture container such that the first surface is proximal andin spaced relation to a bottom surface of the cell culture container,thereby defining within the cell culture container a lower volumecomprising the at least one tumor cell type and an upper volumecomprising a second surface of the porous membrane, wherein the shearstress is applied upon the second surface of the porous membrane in theupper volume of the container.
 4. The method of claim 1, furthercomprising plating endothelial cells on the second surface of the porousmembrane and applying the shear stress upon the plated endothelialcells, wherein the at least one tumor cell type is plated on the firstsurface of the porous membrane.
 5. The method of claim 1, wherein uponapplication of the shear stress, a change in the level or localizationof a marker of the tumor microenvironment in the at least one tumor celltype or a change in the level of a marker of the tumor microenvironmentin the culture medium, as compared to the level or localization of themarker in the at least one tumor cell type or the level of the marker inthe culture medium in the absence of application of the shear stressconfirms mimicking of the tumor microenvironment.
 6. The method of claim3, wherein the cell culture container further comprises inlets andoutlets within the portions of the cell culture container defining theupper and lower volumes.
 7. The method of claim 3, further comprisingperfusing culture medium into and out of the upper volume and perfusingculture medium into and out of the lower volume.
 8. The method of claim1, wherein the at least one tumor cell type comprises cells derived froma carcinoma, a sarcoma, a lymphoma, an adenosquamous carcinoma, a mixedmesodermal tumor, carcinosarcoma, a teratocarcinoma, or a combinationthereof.
 9. The method of claim 1, wherein the at least one tumor celltype is derived from a tumor of connective tissue, a tumor ofendothelium or mesothelium, a tumor of lymphoid tissue, a tumor ofmuscle, a tumor of an epithelial tissue, a tumor of a neural tissue, atumor of the amine precursor uptake and decarboxylation (APUD) system, atumor of a neural crest-derived cell, a gonadal tumor, or a combinationthereof.
 10. The method of claim 1, wherein the at least one tumor celltype comprises primary tumor cells obtained from a subject by biopsy,tumor resection, blood draw, or a combination thereof.
 11. The method ofclaim 4, wherein: the endothelial cells comprise microvascularendothelial cells, macrovascular endothelial cells, endothelialprogenitor cells, or a combination thereof; the endothelial cellscomprise endothelial cells derived from a tumor, or the endothelialcells comprise endothelial cells derived from an organ or tissue inwhich a tumor resides; the endothelial cells comprise endothelial cellsderived from lung, breast, colon, rectum, prostate, bladder, bone,pancreas, liver, bile duct, ovary, testis, uterus, placenta, brain,cartilage, smooth muscle, striated muscle, a membranous lining of a bodycavity, fibrous tissue, blood vessel, lymph vessel, lymph node, adiposetissue, neurogenic connective tissue of the brain, kidney, pituitarygland, parathyroid, thyroid, bronchial lining, adrenalmedulla, stomach,large intestine, small intestine, carotid body, chemoreceptor system,skin, gall bladder, or a combination thereof; or the endothelial cellscomprise cells derived from inducible pluripotent stem cells (iPSC). 12.The method of claim 2, wherein the at least one stromal cell typecomprises fibroblasts, immune cells, pericytes, inflammatory cells, or acombination thereof.
 13. The method of claim 2, further comprisingmixing the at least one stromal cell type with the at least one tumorcell type prior to plating.
 14. The method of claim 2, wherein themethod comprises sequentially plating the at least one tumor cell typeand the at least one stromal cell type.
 15. The method of claim 1,wherein the method comprises culturing the at least one tumor cell typein the substantial absence of exogenously added extracellular matrix.16. The method of claim 14, comprising plating the at least one stromalcell type and subsequently plating the at least one tumor cell type onthe plated stromal cell type.
 17. The method of claim 1, wherein the atleast one tumor cell type comprises tumor cells derived from a humanizedmouse bearing a tumor derived from a human subject.
 18. The method ofclaim 17, wherein the humanized mouse comprises a non-obese diabeticsevere combined immunodeficiency (NOD SCID) mouse, aNOD/Shi-scid/IL-2Rγnull (NOG) mouse, or a NOD SCID IL-2Rγ knockout (NSG)mouse.
 19. The method of claim 12, wherein the at least one stromal celltype comprises the fibroblasts, the fibroblasts comprising human lungfibroblast cell line Hs888Lu.
 20. The method of claim 1, wherein theculture medium comprises sera, blood, blood cells, a blood component,immune cells, conditioned culture medium, or a combination thereof. 21.The method of claim 20, wherein the sera, blood, blood cells, bloodcomponent, or immune cells are derived from a human or other animal. 22.The method of claim 21, wherein the animal is a mouse, rat, guinea pig,hamster, rabbit, cat, dog, monkey, cow, pig, horse, goat, sheep, bird,or fish.
 23. The method of claim 20, wherein the immune cells comprise Bcells, dendritic cells, granulocytes, innate lymphoid cells,megakaryocytes, monocytes, macrophages, natural killer cells, T cells,thymocytes, or a combination thereof.
 24. The method of claim 20,wherein the blood cells comprise platelets, red blood cells, or acombination thereof.
 25. The method of claim 20, wherein the bloodcomponent comprises a clotting factor, a lipoprotein, a triglyceride, ora combination thereof.
 26. The method of claim 20, wherein theconditioned culture medium comprises conditioned culture medium from aculture comprising tumor cells, a culture comprising endothelial cells,a culture comprising a stromal cell type, or a combination thereof. 27.The method of claim 1, wherein the shear stress applied upon the atleast one tumor cell type is about 0.1 dynes/cm² to about 200 dynes/cm².28. The method of claim 27, wherein the shear stress applied upon the atleast one tumor cell type is about 0.1 dynes/cm² to about 100 dynes/cm².29. The method of claim 1, wherein the shear stress is applied at a rateof about 1 sec⁻¹ to about 1000 sec⁻¹.